Early during the course of HIV-1 infection there is a profound loss of CD4+ T cells which is particularly devastating within the gut associated lymphoid tissue (GALT). CD4+CCR6+ T cells are highly susceptible to infection compared to CCR6¯ cells and this subset is preferentially depleted from both GALT and the peripheral blood. CCR6+ cells are crucial for maintaining gut immune function and homeostasis. In animal models, the absence of CCR6 or either of its ligands, macrophage inflammatory protein-3α (MIP-3α) or human beta defensin 2 (hBD2), results in impaired gut immune responses and defects in lymphoid architecture. Depletion of CCR6+ T cells from the GALT may further contribute to immune dysfunction in the gut during HIV-1 infection. Decreased immune function and compromised barrier protection leaves the gut more susceptible to microbial pathogens and microbial translocation across the gut lumen which can trigger immune activation, a strong predictor of disease progression. Therefore, therapeutic strategies that target CCR6+ T cell subsets are needed. We have identified a novel CCR6-dependent mechanism of HIV-1 inhibition mediated by hBD2. The anti-HIV-1 attributes of hBD2 combined with broad antimicrobial activity against gram-negative bacteria and fungi make this endogenous peptide an attractive candidate for potential application as a microbicide. Our previous work demonstrated that treatment of HIV-1 virions with hBD2 reduces infectivity. Here, we report an additional mechanism of inhibition that is CCR6-dependent and occurs after virus entry at an early stage of infection either prior to or during reverse transcription. This post-entry inhibition requires induction of the host restriction factor, apolipoprotein B mRNA editing enzyme-catalytic polypeptide-like 3G (APOBEC3G). In our proposed model, hBD2 inhibits HIV-1 by two distinct mechanisms: inactivation of virions and post-entry mediated by CCR6. Identification of this unique CCR6-mediated post-entry inhibition contributes to our understanding of pathways that underlie intrinsic immunity to HIV-1 and may ultimately guide development of effective therapeutics to selectively target and protect CCR6+ cells.