Women acquire human immunodeficiency virus (HIV) mainly through sexual intercourse. However, low transmission rates per sexual act indicate that local immune mechanisms contribute to HIV prevention. Neutrophils represent 10-20% of the genital immune cells in healthy women. Neutrophils mediate mucosal protection against bacterial and fungal pathogens through different mechanisms, including the release of neutrophil extracellular traps (NETs). NETs are DNA fragments associated with antimicrobial granular proteins. Despite neutrophil abundance and central contributions to innate immunity in the genital tract, their role in protection against HIV acquisition is unknown. We found that stimulation of human genital neutrophils with HIV viral-like particles (HIV-VLPs) induced NET release within minutes of viral exposure, through reactive oxygen species-independent mechanisms that resulted in immediate entrapment of HIV-VLPs. Incubation of infectious HIV with pre-formed genital NETs prevented infection of susceptible cells through irreversible viral inactivation. HIV inactivation by NETs from genital neutrophils could represent a previously unrecognized form of mucosal protection against HIV acquisition.