Background: Hepatitis E virus (HEV)genotype 3 is endemic in Europe, and the infection is mostly subclinical or acute and self-limiting. However, persistent infection is described among HIV-infected individuals. The prevalence of antibodies against HEV (anti-HEV)among HIV-infected persons varies geographically and is unknown in Denmark. Rates of co-infection with HEV among HIV-infected individuals in Denmark over three decades, from the early 1980s to 2013, were investigated. Methods: A total of 2506 HIV-infected persons were investigated from two cohorts followed at Hvidovre Hospital, Denmark. Blood samples were tested retrospectively for anti-HEV, including samples from 2216 persons who were enrolled in a prospective clinical cohort and followed between 1995 and 2013, as well as samples from 290 persons from a historical cohort followed between 1980 and 1994. For anti-HEV seroconverting individuals, serial samples were tested for HEV RNA. Factors associated with anti-HEV status were explored using multivariable logistic regression analysis. Results: The overall HEV seroprevalence rates were stable during the 1980s, 1990s, and 2000–2013 (23.1%, 22.9%, and 23.7%, respectively). In all decades, rates of anti-HEV increased with older age, and anti-HEV seropositivity was associated with older generations, HIV risk group, and geographic origin. Persistent HEV infection was not detected in any of 57 individuals with anti-HEV seroconversion. Conclusions: HEV seroprevalence rates were stable in HIV-infected individuals from the early 1980s to 2013. Rates increased with age. No evidence of persistent HEV infection was detected. Infection with HEV is frequent, but persistent HEV infection is rare among HIV-infected individuals.