The study examined trends in injection risk behaviors among people who inject drugs (PWIDs) and assessed the impact of harm reduction programs in Ukraine during 2007-2013. We performed a secondary analysis of the data collected in serial cross-sectional bio-behavioral surveillance surveys administered with PWIDs in Ukraine in 2007, 2008, 2011, and 2013. Using data from 14 Ukrainian cities, we assessed short-term trends in injection risk behaviors with the Cochran-Armitage test for trend and multivariable logistic regression models, adjusted for age, sex, region, marital status, education level, occupation, age at injection drug use initiation, experience of overdose, and self-reported HIV status. The overall test for trend indicated a statistically significant decrease over time for sharing needle/syringe during the last injection (p < 0.0001), sharing needle/syringe at least once in the last 30days (p < 0.0001), and using a common container for drug preparation (p < 0.0001). The prevalence of injecting drugs from pre-loaded syringes was high (61.0%) and did not change over the study period. After adjusting for all significant confounders and comparing to 2007, the prevalence of sharing needle/syringe during the last injection was unchanged in 2008 (OR = 1.06, 95% CI = 0.92, 1.21), and declined in 2011 (OR = 0.18, 95% CI = 0.15, 0.22) and 2013 (OR = 0.17, 95% CI = 0.14, 0.21). Sharing needles/syringes in the last 30days significantly decreased when compared to that in 2007 (2008: OR = 0.81, 95% CI = 0.74, 0.89; 2011: OR = 0.43, 95% CI = 0.38, 0.47; and 2013: OR = 0.31, 95% CI = 0.27, 0.35). The prevalence of using common instruments for drug preparation also decreased compared to that in 2007 (2008: OR = 0.88, 95% CI = 0.85, 0.91; 2011: OR = 0.85, 95% CI = 0.85, 0.90; and 2013: OR = 0.74, 95% CI = 0.71, 0.76). The observed reduction in the prevalence of injection risk behavior over time is encouraging. Our findings suggest that prevention programs in Ukraine have positive impact and provide support for governmental expansion of these programs.