In the absence of selection, the ability of bacteria to express resistance to antibiotics often results in reduced fitness in vitro and in vivo.  However, due to the ability of drug efflux pumps to also export host antimicrobials (e.g., antimicrobial peptides), we hypothesized that the expression of efflux pump genes could promote bacterial growth in vivo.  Using a female mouse model of lower genital tract infection, we tested whether levels of the mtrCDE-encoded efflux pump, which is known to export host antimicrobials, possessed by the strict human pathogenic Neisseria gonorrhoeae influences bacterial survival and fitness during infection.  The mtrCDE operon (and > 50 other genes) is negatively regulated by the MtrR repressor and loss of MtrR was found to increase bacterial resistance to host  antimicrobials and enhance in vivo fitness.  In contrast, the mtrCDE operon (and > 40 other genes) is transcriptionally activated by MtrA and loss of MtrA was found to result in reduced fitness in vivo; this fitness cost could, however, be reversed by second site mutations in mtrR.  We propose that the MtrC-MtrD-MtrE efflux pump is a gonococcal virulence factor and its level of expression, modulated by MtrR and MtrA, determines both levels of resistance to host antimicrobials and in vivo fitness.