We present data from an observational cohort study on human immunodeficiency virus (HIV) prevention and control measures in prisons in Estonia to assess the potential for HIV transmission in this setting. HIV testing and retesting data from the Estonian prison health department were used to estimate HIV prevalence and incidence in prison. Since 2002, voluntary HIV counselling and testing has routinely been offered to all prisoners and has been part of the new prisoners health check. At the end of 2012, there were 3,289 prisoners in Estonia, including 170 women: 28.5% were drug users and 15.6% were infected with HIV. Of the HIV-positive inmates, 8.3% were newly diagnosed on prison entry. In 2012, 4,387 HIV tests (including retests) were performed in Estonian prisons. Among 1,756 initially HIV-negative prisoners who were in prison for more than one year and therefore tested for HIV twice within 12 months (at entry and annual testing), one new HIV infection was detected, an incidence of 0.067 per 100 person-years (95% confidence interval (CI): 0.025–5.572). This analysis indicates low risk of HIV transmission in Estonian prisons. Implementation of HIV management interventions could impact positively on the health of prisoners and the communities to which they return.