HIV infects macrophages and microglia in the central nervous system (CNS). Mechanisms that enhance HIV macrophage/microglial tropism are not well understood. Here, we identify an HIV Env variant in the V4 region of gp120, Asp 386 (D386), that eliminates an N-linked glycosylation site at position 386, enhances viral replication in macrophages, and is present at a higher frequency in AIDS patients with HIV-associated dementia (HAD) compared with non-HAD patients. D386 enhances HIV entry and replication in macrophages but not in microglia or peripheral blood mononuclear cells, possibly due to differential glycosylation in these cell types. A D386N mutation in the UK1br Env, which restores the N-linked glycan site, reduced neutralization sensitivity to the IgG1b12 (b12) monoclonal antibody, which recognizes a conserved neutralization epitope that overlaps the CD4 binding site. Molecular modeling suggested that loss of the glycan at position 386 increases exposure of the CD4 and b12 binding sites on gp120. Loss of a glycan at 386 was more frequent in Envs from HAD patients (26%; n=185) compared with non-HAD patients (7%; n=99; p<0.001). The most significant association of these Env variants with HAD was in blood or lymphoid tissue rather than brain. These findings suggest that increased exposure of the b12 epitope overlapping the CD4 binding site via elimination of a glycan at position 386 is associated with enhanced HIV macrophage tropism, and provide evidence that determinants of macrophage and microglia tropism are overlapping but distinct.