Background: Female who inject drugs are exposed to greater social disapproval, stigmatization, and report a higher risk of unsafe injection practices compared with their male counterparts. Gender differences in injection practices among people who inject drugs (PWID) in Latin America remain understudied. Objectives: This study aimed to examine gender differences in injection practices among PWIDs in Colombia. Materials and Methods: Using respondent-driven sampling, N=1081 participants (14% female; mean age: 26 y) from 5 Colombian cities completed the Pan American Health Organization’s Behaviors of High Risk Drug Consumers Survey. A binary logistic regression model was used to examine first and last injection practices and risk injection behaviors among PWIDs. Results: Female presented high odds of receiving assistance by their sexual partners during the first and last injection episode [adjusted odds ratio (AOR)=8.3, confidence interval (CI)=14.0-16.7; AOR=2.06, CI=1.1-3.7]. Also, female had at least 2 times higher odds of engaging in unsafe injecting practices including sharing drug mix and using drug in a capsule or ready-to-use vial (AOR=2.8, CI=1.4-5.5; AOR=3.4, CI=1.5-7.7) and had lower odds of acquiring the drug to self-inject (AOR=0.4, CI=0.2-0.9) during the last injection episode. Conclusions: High dependence on sexual partner and risky injection behaviors among female who inject drugs were identified. Study findings contextualized with gender vulnerabilities in Colombia are fundamental for prevention/intervention efforts aimed at helping this at-risk population in the country and in Latin America.