OBJECTIVES: Multidrug-resistant, vancomycin-nonsusceptible Staphylococcus capitis is an emerging cause worldwide of late-onset sepsis (LOS) in preterm neonates. The pathophysiology and risk factors for S.~capitis-related LOS are poorly understood, but we hypothesized that S.~capitis LOS follows translocation from the gut microbiota rather than catheter invasion. The objective of this study was to investigate the risk factors of S.~capitis LOS and gut colonization. METHODS: We conducted a prospective single-centre cohort study of patients hospitalized in a tertiary-care unit (Lyon, France) from June 2011 to January 2012. S.~capitis gut colonization was determined weekly from stool cultures. The determinants of gut colonization and LOS were established by multivariate Cox proportional hazards models. RESULTS: Eighty-three (36.2%) of 229 patients had S.~capitis-positive stool culture, and 28 (12.2%) developed S.~capitis LOS during hospitalization. Independent risk factors for S.~capitis LOS included prior administration of vancomycin independent of a previous LOS episode (hazard ratio 6.44, 95% confidence interval 2.15-19.3, p 0.001) and low birth weight (hazard ratio 0.72 per 100~g increase, 95% confidence interval 0.55-0.95, p 0.02). The prior administration of vancomycin was also an independent risk factor for S.~capitis colonization (hazard ratio 3.45, 95% confidence interval 2.07-5.76, p \textless0.001), particularly in the first week of life and in noncolonized neonates. CONCLUSIONS: Neonates treated with vancomycin are at a higher risk of LOS caused by vancomycin-nonsusceptible S.~capitis. The use of vancomycin in neonates must urgently be optimized to limit the selection of vancomycin-nonsusceptible strains, for which alternative antibiotics are lacking.