HIV is a pandemic disease, and many cellular and systemic factors are known to alter its infectivity and replication. Earlier studies had suggested that anemia is common in HIV-infected patients; however, higher iron was also observed in AIDS patients prior to the introduction of antiretroviral therapy (ART). Therefore, the relationship between iron and viral infection is not well delineated. To address this issue, we altered the levels of cellular iron in primary CD4(+) T cells and showed that higher iron is associated with increased HIV infection and replication. In addition, HIV infection alone leads to increased cellular iron, and several ART drugs increase cellular iron independent of HIV infection. Finally, HIV infection is associated with increased serum iron in HIV-positive patients regardless of treatment with ART. These results establish a relationship between iron and HIV infection and suggest that iron homeostasis may be a viable therapeutic target for HIV.