Background: Prisoner’s addiction is one of the major problems in many countries which imposes very high medical costs and social harm to communities. This study investigated the pattern of substance use and related factors in male prisoners in one of the prisons in southeastern Iran.Methods: This cross-sectional study was carried out in 2016. The study population was inmates of a prison in southeast Iran. Sampling was carried out randomly according to the list of prisoners. Data were collected using a form and were analyzed with statistics software SPSS.Findings: More than four-fifths (75.3%) of the subjects consumed at least one substance (alcohol, tobacco and other drugs), 74.4% were smoking, 73.2% used a narcotic substance, and about one-fifth (19.3%) reported drinking alcohol. With a frequency of 62.0%, opium was the most frequently utilized narcotic substance. Poppy juice (31.6%), cannabis (29.8%), crystal (16.9%) and tramadol (16.9%) were the next frequent substances used. A percentage of 41.5% subjects reported using two or more drugs. A percentage of 80.7% subjects reported substance use among their friends, 39.2% by siblings and 37.2% by father. Regression analysis showed predictor variables of substance use were education, substance use by prisoner before being imprisoned, substance use by father, friends and siblings.Conclusion: This study showed a remarkable prevalence of substance use in prisons, which was more than general population. Therefore, it is necessary to consider alternative penalties of imprisonment due to the factors associated with substance use. Screening of people at high risk for substance use should be considered on admission to prison, and primary prevention measures should be focused on them.