Neisseria gonorrhoeae is a common bacterial sexually transmitted infection. Like all Gram-negative bacteria, the outer membrane of the gonococcus is rich in endotoxin, a known ligand for Toll-like receptor (TLR)4. However, the role of endotoxin and that of its cognate receptor TLR4 in the mucosal response to acute gonococcal infection in the genital tract of women is unclear. To test this, we examined the course of infection after vaginal inoculation of N. gonorrhoeae in mice carrying the Lps(d) mutation in Tlr4, which renders them unresponsive to endotoxin. Although there was no difference in the duration of colonization, Lps(d) mice had a significantly higher peak bacterial burden which coincided with a massive polymorphonuclear cell influx and concomitant upregulation of a subset of inflammatory cytokine and chemokine markers. Notably, infected Lps(d) mice showed a decrease in interleukin-17, suggesting that Th17 responses are more dependent on TLR4 signaling in vivo. Defective polymorphonuclear cell-mediated and complement-independent serum killing of gonococci in Lps(d) mice was also observed and may account for the increased bacterial burden. This is the first in vivo evidence that TLR4-regulated factors modulate early inflammatory responses to gonococcal infection in the female reproductive tract and control bacterial replication.