We studied mechanisms driving gender differences in HIV incidence among 651 women and men who inject drugs (PWID) in Tijuana, Mexico, hypothesizing that sex work will mediate the association between female gender and HIV incidence. Of 43 HIV seroconversions occurring between 2011 and 2018, 8.8% were among females and 5.2% among males. HIV incidence density was significantly higher among females versus males (1.75 per 100 person years [PY], 95% CI 1.16-2.66, vs. 0.95 per 100 PY, 95% CI 0.62-1.47). Factors significantly associated with HIV seroconversion were: sex work (adjusted hazard ratio [aHR] = 2.25, 95% CI 1.05-4.80); methamphetamine injection (aHR = 2.30, 95% CI 1.12-4.73); and methamphetamine and heroin co-injection in the past six months (aHR = 2.26, 95% CI 1.23-4.15). In mediation analyses, sex work mediated a substantial proportion (84.3%) of the association between female gender and HIV incidence. Interventions should target female PWID who engage in sex work to reduce gender-related disparities in HIV incidence.