The aim of this study was to investigate the relationship between nasal and rectal Staphylococcus aureus carriage in intensive care unit (ICU) patients and the occurrence of ICU-acquired infections related to S. aureus carriage. Three hundred and ninety-five patients admitted in ICU were screened for S. aureus nasal and rectal carriages and followed to record S. aureus infections during their stay. S. aureus strains were genotyped by arbitrarily primed PCR, spa-typing, microarray and whole genome sequencing. At ICU admission, 112 of 363 (30.9%) patients carried S. aureus including 61 (16.8%) exclusive nasal carriers, 40 (11.0%) combined nasal and rectal carriers and 11 (3.0%) exclusive rectal carriers. The 152 S. aureus isolates from nasal and rectal swabs belonged to 19 clonal complexes (CCs). Patients colonized in both nose and rectum harboured different strains in at least 40% of cases according to arbitrarily primed PCR data. Nasal carriers of CC5 S. aureus had an increased risk of rectal carriage (RR = 1.85, P < .05). S. aureus nasal and rectal carriage was a risk factor of S. aureus ICU-acquired infection (RR = 4.04; 95%CI [1.38-11.76]). Incidence rates of endogenous ICU-acquired infections in exclusive nasal carriers, exclusive rectal carriers and in both nasal and rectal carriers were 0.08 (5/61), 0.09 (1/11) and 0.03 (1/40), respectively (p = 0.47). Rectal swabbing increased the detection of S. aureus carriage and revealed an important diversity of S. aureus strains in ICU patients. Further studies are needed to understand how S. aureus rectal carriage increases the risk of endogenous ICU-acquired infections.