Hepatitis C virus (HCV) infection is the most common blood-borne viral infection in haemodialysis. It causes significant morbidity and long-term mortality. Practice of universal precautions has been reported to be sufficient to prevent HCV seroconversion in dialysis units. However, the seroconversion rate remains very high in many dialysis units. A previous study from 1995 to 1998 at our own hospital without isolation showed that nosocomial transmission is the major cause of HCV seroconversion. The present study was therefore conducted with the aim to study the impact of isolation on HCV seroconversion. In this prospective cohort study, with non-probability consecutive sampling, patients with HCV infection were dialysed in an isolated room. In addition, standard universal precautions were practiced. HCV seroconversion rate was compared with the previous study. All patients with end-stage kidney disease (ESKD) admitted to our hospital for renal replacement therapy were included in the present study. At the time of admission, HCV screening was done. All anti-HCV-positive patients were dialysed in an isolated room. While on maintenance haemodialysis, all patients were monthly tested for anti-HCV, aspartate aminotransferase and alanine aminotransferase. Any patient who had HCV seroconversion was transferred to an isolated room for maintenance haemodialysis. Patients with HCV infection were managed by further testing for HCV-RNA and liver biopsy. Every patient who ultimately received renal transplantation at our hospital was also tested for HCV just prior to renal transplantation as well as 3 months after renal transplantation. HCV infection was diagnosed by detecting anti-HCV antibodies using an ELISA-based third-generation diagnostic test kit. Serum bilirubin, aspartate aminotransferase and alanine aminotransferase were assayed using standard laboratory techniques. From March 2003 to February 2006, 1,417 patients were admitted for haemodialysis in our unit. Of these 1,077 (76%) had ESKD. Mean age of patients was 42.47 ± 16.2 (14–94) and 70.39% were males. Patients with ESKD had had more dialysis sessions (10.9 ± 39.5 vs. 4.4 ± 5.95, p = 0.009), more blood transfusions and more pre-existing HCV infections (4.72 vs. 1.5%, p = 0.009) than patients with acute renal failure. Of the ESKD patients, 65.7% were discharged, 9.47% died, 1.85% were shifted to chronic ambulatory peritoneal dialysis and 22.46% patients received renal transplantation. Of the patients who received renal transplantation, HCV seroconversion was detected in 2.75%. In the previous study without isolation practices, the HCV seroconversion rate in transplanted patients was 36.2%. The hazard of HCV seroconversion was 0.97 (95% CI 0.93–1.02, p = 0.2) for each additional dialysis and 1.09 (95% CI 0.88–1.36, p = 0.37) for each additional blood transfusion. The study concludes that isolation of HCV-infected patients during haemodialysis significantly decreases the HCV seroconversion rate.