The success of an HIV vaccine will require induction of a protective immune response in the most at-risk populations. The increased incidence of HIV infection in high-risk populations is assumed to be primarily the result of more frequent exposure to the virus or a greater inoculum of the virus; however, underlying variations in immune homeostasis may also contribute to HIV susceptibility and potentially impact vaccine responses and those required for protection. As an effective humoral immune response is likely to be a critical component of a protective HIV vaccine, we evaluated the steady-state phenotypic profile of peripheral blood B cells by flow cytometry from participants in the HIV Vaccine Trials Network (HVTN) 203 Phase 2a HIV vaccine trial considered to be at higher risk and lower risk for HIV acquisition. Overall, high-risk participants exhibited increased frequency of unswitched IgM memory and activated switched IgD-CD95+ memory B cells than low-risk participants. Most (93%) of the high-risk male participants were men who have sex with men who engaged in high-risk sexual behavior. High-risk males had a significantly increased frequency of CXCR3+ IgD-CD95+ B cells than low-risk males. These results suggest that high-risk populations have altered B cell homeostasis. The increased frequency of activated and memory B cells may suggest increased immune activation in high-risk populations, which may contribute to possible differential responses to HIV vaccine strategies. Copyright Mary Ann Liebert, Inc 2016.