Objectives Studies showed that men who have sex with men (MSM), including those using pre-exposure prophylaxis (PrEP), are at increased risk of hepatitis C virus (HCV) infection. We evaluated HCV prevalence and incidence, along with their associated determinants, in a cohort of PrEP-using individuals in the Netherlands. Methods In 2019, the Netherlands launched a 5-year national programme that offers subsidised PrEP to eligible individuals. We used prospectively collected data from individuals registered in this programme between 2019 and 2022. Individuals underwent annual testing for HCV antibodies and additional HCV-RNA testing when antibodies were present. We calculated the prevalence of past/current HCV infection at first visit and overall incidence rate (IR) during follow-up. Univariable logistic and Poisson regression models were used to identify determinants associated with past/current prevalent or incident HCV infection, respectively. Behavioural factors referred to those occurring in the previous 6 months. Results A total of 10 563 (n=10 319, 97.7% MSM) were included. At first visit, 66 of 10 563 (0.6%) had a past/current HCV infection, which was associated with older age [odds ratio (OR) per 10 years=1.57, 95% confidence interval (CI)=1.31 to 1.88], the use of PrEP before first visit (OR=3.03, 95% CI=1.79 to 5.13), receptive condomless anal sex (CAS) (OR=2.73, 95% CI=1.25 to 5.98), chemsex (OR=2.44, 95% CI=1.49 to 3.99) and injecting drug use (IDU) (OR=6.61, 95% CI=2.35 to 18.61). Among 9851 individuals contributing to 17 150 person-years (PYs) of follow-up, 64 incident HCV infections (IR=0.37 per 100 PYs, 95% CI=0.29 to 0.48) were identified. Factors associated with incident HCV infection were receptive CAS [incidence rate ratio (IRR)=2.59, 95% CI=1.12 to 6.02], chemsex (IRR=1.78, 95% CI=1.06 to 2.98), sexually transmitted infection diagnosis (IRR=2.30, 95% CI=1.23 to 4.31) and IDU (IRR=6.15, 95% CI=2.20 to 17.18). Conclusions Past/current prevalence and incidence of HCV were low among individuals in ...