Male genitourinary tract (MGT) bacterial infections are considered responsible for 15% of male infertility, but the mechanisms underlying decreased semen quality are poorly known. We evaluated in vitro the effect of strains of Gram-negative uropathogenic species (two E.coli strains, three K. pneumoniae strains, P. aeruginosa and E. cloacae) on motility, viability, mitochondrial oxidative status, DNA fragmentation and caspase activity of human spermatozoa. All strains, except P. aeruginosa, reduced significantly sperm motility, with variable effects. Sperm Immobilizing Factor (SIF) was largely responsible for deteriorating effects on sperm motility of E. coli strains since they were completely reverted by knockout of SIF coding recX gene. Sequence alignment for RecX showed the presence of high homologous sequences in K. pneumoniae and E. cloacae but not in P. aeruginosa. These results suggest that, in addition to E.coli, other common uropathogenic Gram-negative bacteria affect sperm motility through RecX products. In addition to sperm motility, the E. coli strain ATCC 35218 also affected sperm viability, and induced caspase activity, oxidative stress and DNA fragmentation suggesting an interspecies variability in the amount and/or type of the produced spermatotoxic factors. In general, our results highlight the need for a careful evaluation of semen infections in the diagnostic process of the infertile man.