Although anemia is common during HIV infection, it is unclear whether potent antiretroviral therapy would improve or worsen anemia. We conducted a study to examine the impact of highly active antiretroviral therapy (HAART) on anemia in a cohort of HIV-positive injection drug users (IDUs) in Baltimore, Maryland. At baseline, the overall prevalence of anemia was 40%. During mean follow-up of one year, among 102 subjects who received HAART, there was a mean increase in hemoglobin of 3.6 ± 1.7 g/L (p = .04) and a mean decrease in log10 plasma HIV load of 0.78 ± 0.17 copies/ml (p <.0001). Among 103 control subjects who were not receiving antiretroviral medications, there was a mean decrease in hemoglobin of 4.2 ± 1.1 g/L (p <.0003) and mean increase in log10 plasma HIV load of 0.25 ± 0.06 copies/ml (p <.0002). Multivariate analysis using mixed linear models showed that HAART was associated with an increase of hemoglobin of 0.223 g/L per month (p <.0001) after adjusting for body mass index, opportunistic infections, and gender. HAART was associated with an improvement in anemia, and potential mechanisms that may be involved include a reduction in opportunistic infections and the anemia of chronic disease and an improvement in nutritional status.