Du Plessis left the field in the Springboks’ 31-28 victory over England last Saturday after injuring his hamstring and knee.
He went for scans in Padova on Monday evening.
"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 Springbok team doctor Craig Roberts.
Willie le Roux, who injured his cheek bone against England, also went for scans on Monday evening, but the results were good and he has been cleared of any fractures.
"We’ll manage Willie during training this week but he should be available to face Italy," said Roberts.
Golden Lions prop Ruan Dreyer will join the Springboks in Padova on Tuesday.
He was called up after the injury concerns over Du Plessis.