American Gatlin, bronze medallist at the London Games after a four-year doping ban, ran 9.94 seconds to edge Bolt by 0.01 in a race that showed the Jamaican has yet to fully recover from the hamstring injury that dogged him earlier in the season.
Bolt's previous defeat over the distance was at last year's Jamaican championships when fans and media were stunned as he was beaten by Yohan Blake only for the world record holder to scorch to the second-fastest time on record of 9.63 in the Olympic final a few weeks later.
The 26-year-old Bolt got a good start in Rome but Gatlin, who also won in Doha and Eugene this season, had moved ahead by halfway and his rival never looked like making up the ground.
No hamstring problems during Rome defeat for Bolt
"That was ridiculous - a perfect start and then I just cropped off. I think it was the perfect start that threw my game off," six-times Olympic gold medallist Bolt told reporters.
"At the end it just wasn't me - at least I got under 10 seconds," he added, referring to his only other race over 100 metres this season when he ran 10.09 in the Cayman islands last month.
"For me it is just going through the season, putting things together for the world championships (in Moscow). The season is still very early."
Women's Olympic 200 metres champion Allyson Felix also suffered a surprise defeat when she was left trailing down the home straight by Ivory Coast's Murielle Ahoure who won in a national record of 22.36.
Felix, easily identifiable in bright pink shin socks and matching spikes, ran a poor bend and lacked her usual fluidity as she raced to the line in 22.64.
"I am very excited after my victory and the national record is a big bonus for me," Ahoure said.
"I was not worried about having strong opponents next to me, it will be the same at the world championships, where I hope my training will pay off. That is what we are working for."
A number of world leading times for the year were recorded on the track, including Amantle Montsho who beat her own 400 metres best for 2013 by running 49.87.
"I am very happy with my time today. I wasn't thinking of the world championships when I ran," Montsho said before rushing off to fly back to Botswana.
Ethiopia's Yenew Alamirew smashed the year's best 5,000 metres time, set by Kenyan Edwin Soi at the Diamond League meet in Eugene last week, by almost 10 seconds when he ran 12:54.95.
Soi, who beat Olympic champion Mo Farah when winning in Eugene, could only manage fifth in 13:02.54 this time.
Mohammed Aman showed he was likely to be Olympic champion David Rudisha's main challenger over 800 metres this year with a world leading 1:43.61.
"I expected to run a good time because my previous races and training were good," said the Ethiopian teenager, who is the only man to beat Rudisha in the last two years.
"Who am I afraid of? Who could beat me? We will see..." he added.
Croatia's Olympic champion Sandra Perkovic overcame illness to comfortably win the women's discus with 68.25 metres, nearly four metres more than nearest rival Cuba's Yarelis Barrios.
1. Justin Gatlin (U.S.) 9.94
2. Usain Bolt (Jamaica) 9.95
3. Jimmy Vicaut (France) 10.02
1. LaShawn Merritt (U.S.) 44.96
2. Yousef Ahmed Masrahi (Saudi Arabia) 45.24
3. Joshua Mance (U.S.) 45.26
1. Mohammed Aman (Ethiopia) 1:43.61
2. Pierre-Ambroise Bosse (France) 1:43.91
3. Andre Olivier (South Africa) 1:44.37
1. Yenew Alamirew (Ethiopia) 12:54.95
2. Hagos Gebrhiwet (Ethiopia) 12:55.73
3. Isiah Kiplangat Koech (Kenya) 12:58.85
Men's 110m Hurdles
1. Sergey Shubenkov (Russia) 13.20
2. Pascal Martinot-Lagarde (France) 13.31
3. Balazs Baji (Hungary) 13.44
Men's 400m Hurdles
1. Johnny Dutch (U.S.) 48.31
2. Javier Culson (Puerto Rico) 48.36
3. Mamadou Kasse Hanne (France) 48.56
Men's Pole Vault
1. Raphael Holzdeppe (Germany) 5.91
2. Renaud Lavillenie (France) 5.86
3. Malte Mohr (Germany) 5.86
Men's Triple Jump
1. Christian Taylor (U.S.) 17.08
2. Daniele Greco (Italy) 17.04
3. Teddy Tamgho (France) 17.01
Men's Shot Put
1. David Storl (Germany) 20.70
2. Cory Martin (U.S.) 20.54
3. Dylan Armstrong (Canada) 20.29
1. Murielle Ahoure (Cote D'Ivoire) 22.36
2. Allyson Felix (U.S.) 22.64
3. Ivet Lalova (Bulgaria) 22.78
1. Amantle Montsho (Botswana) 49.87
2. Francena McCorory (U.S.) 50.05
3. Natasha Hastings (U.S.) 50.53
1. Abeba Aregawi (Sweden) 4:00.23
2. Genzebe Dibaba (Ethiopia) 4:01.62
3. Jennifer Simpson (U.S.) 4:02.30
Women's 100m Hurdles
1. Dawn Harper-Nelson (U.S.) 12.65
2. Lolo Jones (U.S.) 12.70
3. Ginnie Crawford (U.S.) 12.90
Women's 3000m Steeplechase
1. Milcah Chemos Cheywa (Kenya) 9:16.14
2. Lydiah Chepkurui (Kenya) 9:18.10
3. Sofia Assefa (Ethiopia) 9:21.24
Women's High Jump
1. Anna Chicherova (Russia) 1.98
1=. Svetlana Shkolina (Russia) 1.98
3. Blanka Vlasic (Croatia) 1.95
4. Emma Green Tregaro (Sweden) 1.92
Women's Long Jump
1. Brittney Reese (U.S.) 6.99
2. Janay DeLoach (U.S.) 6.97
3. Shara Proctor (Britain) 6.91
Women's Discus Throw
1. Sandra Perkovic (Croatia) 68.25
2. Yarelys Barrios (Cuba) 64.41
3. Zinaida Sendriute (Lithuania) 62.85
Women's Javelin Throw
1. Christina Obergfoell (Germany) 66.45
2. Maria Abakumova (Russia) 64.03
3. Sunette Viljoen (South Africa) 63.49
'I want to show people I can do music too and win a Grammy' - Bolt launches reggae album
WADA to review cannabis rule after Richardson ban