ABSTRACTCervicovaginal mucus (CVM) can provide a barrier that precludes HIV and other sexually transmitted virions from reaching target cells in the vaginal epithelium, thereby preventing or reducing infections. However, the barrier properties of CVM differ from woman to woman, and the causes of these variations are not yet well understood. Using high-resolution particle tracking of fluorescent HIV-1 pseudoviruses, we found that neither pH nor Nugent scores nor total lactic acid levels correlated significantly with virus trapping in unmodified CVM from diverse donors. Surprisingly, HIV-1 was generally trapped in CVM with relatively high concentrations ofd-lactic acid and aLactobacillus crispatus-dominant microbiota. In contrast, a substantial fraction of HIV-1 virions diffused rapidly through CVM with low concentrations ofd-lactic acid that had aLactobacillus iners-dominant microbiota or significant amounts ofGardnerella vaginalis, a bacterium associated with bacterial vaginosis. Our results demonstrate that the vaginal microbiota, including specific species ofLactobacillus, can alter the diffusional barrier properties of CVM against HIV and likely other sexually transmitted viruses and that these microbiota-associated changes may account in part for the elevated risks of HIV acquisition linked to bacterial vaginosis or intermediate vaginal microbiota.IMPORTANCEVariations in the vaginal microbiota, especially shifts away fromLactobacillus-dominant microbiota, are associated with differential risks of acquiring HIV or other sexually transmitted infections. However, emerging evidence suggests thatLactobacillus inersfrequently colonizes women with recurring bacterial vaginosis, raising the possibility thatL. inersmay not be as protective as otherLactobacillusspecies. Our study was designed to improve understanding of how the cervicovaginal mucus barrier against HIV may vary between women along with the vaginal microbiota and led to the finding that the vaginal microbiota, including specific species ofLactobacillus, can directly alter the diffusional barrier properties of cervicovaginal mucus. This work advances our understanding of the complex barrier properties of mucus and highlights the differential protective ability of different species ofLactobacillus, withLactobacillus crispatusand possibly other species playing a key role in protection against HIV and other sexually transmitted infections. These findings could lead to the development of novel strategies to protect women against HIV.