We examined the relationship between duration of injecting career and HIV seroconversion among people who inject drugs (PWID) in Vancouver, Canada. Data were derived from HIV-negative PWID enrolled in a prospective cohort study. We employed Kaplan-Meier methods and Cox regression to investigate the effect of length of time since injection drug use initiation on time to HIV seroconversion. In multivariable Cox analysis, duration of injecting career was negatively associated with time to HIV seroconversion (adjusted hazard ratio = 0.82; 95% confidence interval [CI]: 0.69–0.97). Our findings highlight the need for interventions that target individuals who participate in high-risk drug use behaviors.