Central venous catheterization is a common technique to establish rapid and temporary access for hemodialysis. However, it is a known risk factor for Staphylococcus aureus infection and bacteremia. Mupirocin is a topical antibiotic with high in vitro anti-staphylococcal activity. A randomized prospective trial was conducted to assess the effectiveness of mupirocin ointment in the prevention of Staphylococcus aureus skin and catheter colonization, and episodes of bacteremia in 136 end-stage renal disease patients. Of these, 67 received skin disinfection at the venous catheter insertion site with povidone iodine (control group), and 69 received the same treatment followed by application of 2% mupirocin ointment at the cannula site after catheter placement and at the end of each dialysis session. Patients were followed until catheter removal and were monitored for the development of Staphylococcus aureus skin/catheter colonization and episodes of bacteremia. Median duration of catheter use was greater in the mupirocin than in the control group (37 versus 20 d, P < 0.01). Patients in the mupirocin group had a significantly lower rate of Staphylococcus aureus isolation from the pericatheter skin (1.76 per 1000 versus 14.27 per 1000 patient-days, P < 0.001) and from the catheter surface (3.17 per 1000 versus 14.27 per 1000 patient-days, P < 0.001). The proportion of patients with Staphylococcus aureus skin infection at the insertion site was lower in the mupirocin group (4.3% versus 23.9%, P = 0.001). Staphylococcus aureus-associated bacteremia was observed in 17 patients (two in the mupirocin group [0.71 episodes per 1000 patient-days] and 15 in the control group [8.92 per 1000 patient-days], P < 0.001). The hazard ratio of developing Staphylococcus aureus bacteremia was 7.2 (95% confidence interval, 1.6 to 31.6) times greater in patients not receiving mupirocin. Mupirocin applied to the insertion site significantly reduces the risk of Staphylococcus aureus skin and catheter colonization, exit-site infection, and Staphylococcus aureus bacteremia in hemodialysis patients.