Background: HEPATITIS B VIRUS (HBV), HEPATITIS C VIRUS (HCV), and human immunodeficiency VIRUS (HIV) are the three prevalent viral and bloodborne infections worldwide. Considering the similar route of transmission in these infections, their co-infections would be more challenging for health care professionals. Therefore, we investigated the rate of HIV/HBV/HCV co-infection among injection drug users (IDUs) referred to Drop in centers (DICs).Materials and Methods: In this cross-sectional study (2008-2009), IDUs referred to DICs in Isfahan province were evaluated. Venous blood samples were obtained and HBsAg, HBcAb, HCVAb, and HIVAb measured by using enzyme linked immunosorbent assay method. Demographic data and risk factors in patients with HBV/HCV, HIV/HCV, and HIV/HBV co-infections were obtained by a trained social worker using a structured checklist. Data were analyzed using Chi-square test, t-test, and multiple logistic regressions.Results: Totally, 539 IDUs with mean (standard deviation [SD]) age of 35.3 (7.9) were studied. HBV/HCV, HCV/HIV, and HBV/HIV co-infections were presented in 65 (12.1%), 6 (1.1%), and 0 (0%) of IDUs, respectively. All HIV infected IDUs were infected with HCV as well. There was a significant association between HBV/HCV co-infection and behaviors related to sharing needle (odds ratio [OR]=2.06, 95% confi dence interval [CI]; 1.23-3.45) and imprisonment (OR=1.01, 95% CI; 1.04-1.06).Conclusion: According to the results of this study, history of imprisonment and needle sharing were the only adjusted risk factors for HCV/HBV co-infection in IDUs. This might be a warning for national health system and needs to urgent paying attention. It seems that expanded harm reduction strategies can be useful to reduce this co-infection and its mortality and morbidity rate among IDUs.