Aims: The study objectives were to examine trends in prescription opioid (PO) injection and to assess its association with HCV seroconversion among injection drug users (IDUs), accounting for other risk factors.Design and Setting: A prospective cohort study of IDUs was carried out between 2004 and 2009 in Montreal, Canada.Participants and Measurements: 246 HCV-negative IDUs were included in this analysis. Semiannual visits included HCV antibody testing and an interview-administered questionnaire assessing risk behaviours. HCV incidence rate was calculated using the person-time method.Time-updated Cox regression models were conducted to examine predictors of HCV incidence.Findings: The proportion of IDUs reporting PO injection increased from 21% to 75% between 2004 and 2009 (p < 0.001). Of the 246 participants (81.6% male; mean age 34.5 years; mean follow-up time 23 months), 83 seroconverted to HCV (incidence rate: 17.9 per 100 person-years; 95% CI 14.3, 22.1). PO injectors were more likely to become infected if they did not use injection heroin (Adjusted Hazard Ratio (AHR): 2.9 (95% CI: 1.5, 5.5)), whereas the association was not statistically significant for participants who reported using both drugs (AHR: 1.2 (95%CI: 0.6, 2.3). Other independent predictors of HCV incidence were: cocaine injection, recent incarceration, and > 30 injections per month.Conclusion: PO injection has increased rapidly in recent years, and appears to be an important risk factor for HCV acquisition. Our results suggest that the risks related to PO injection may be conditioned by specific drug practices which differ from those of heroin users.