Alpha-defensins, including human neutrophil peptides 1-3 (HNP1-3) and human defensin 5 (HD5), are elevated at the genital mucosa in individuals with sexually transmitted infections (STIs). The presence of STIs is associated with an increased risk of human immunodeficiency virus (HIV) transmission, suggesting there may be a role for defensins in early events of HIV transmission. HD5 has been demonstrated to contribute to STI-mediated increased HIV infectivity in vitro. HNPs exhibit anti-HIV activity in vitro. However, increased levels of HNPs have been associated with enhanced HIV acquisition and higher viral load in breast milk. This study found that HNP1, but not HD5, significantly disrupted epithelial integrity and promoted HIV traversal of epithelial barriers. Linear HNP1 with the same charges did not affect epithelial permeability, indicating that the observed effect of HNP1 on the epithelial barrier was structure dependent. These results suggest a role for HNP1 in STI-mediated enhancement of HIV transmission.