Du Plessis hyper-extended his knee and strained a hamstring in the Springboks' 31-28 victory over England last Saturday and his absence for the weekend was confirmed by team doctor Craig Roberts in a press release from South African Rugby Union (SARU).
"Fortunately Jannie's knee is fine but the scans confirmed our suspicion of a hamstring strain and he will not be able to play against Italy on Saturday," said Roberts.
Le Roux, who injured his cheekbone against England, also went for scans on Monday evening and has been cleared of any fractures.
"We'll manage Willie during training this week but he should be available to face Italy," said Roberts.
Lions prop Ruan Dreyer will join the Boks in Padova on Tuesday as a replacement for Du Plessis.