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Objective. The problem of substance abuse is found all over the world. Abused substances are psychoactive and may drive abusers to engage into unsafe sexual practices and criminal activities. For injecting drugs, the apparatus used often unsterile and shared which predispose the users to blood borne infections. The purpose was to determine the role of substance abuse in transmission of HIV and its magnitude in Mwanza Urban Tanzania. Methods. A cross-sectional study of 774 substance abusers aged 18 years and above was conducted from March to June 2012. Data collection involved use of a questionnaire (Face to face approach) and collection of blood to determine the serological status, while urine samples were used to detect illegal drugs which had been used. Results. Cannabis and alcohol were highly abused 83.0% and 74.2% respectively followed by khat 38.3 %. Heroin smoking was 34.0% and injection was 6.1%. HIV infection was detected in 8.6% of the substance abusers, while Hepatitis B and C were 3.8% and 2.9% respectively and 2.5% Syphilis. Conclusion. HIV infection in substance abusers was higher 8.6% with 6.3% among intravenous drug users. Education on consequences of psychoactive substances and unsafe sex should be given to the substance abusers.