Infection is a powerful mechanism that can lead to sperm damage, deformity and eventually, male infertility. Genital tract infection and inflammation have been associated to 8-35% of male infertility cases. E. coli causes agglutination of human spermatozoa and thus, leading to infertility. This study evaluated if the negative influence of Escherichia coli on the motility of human spermatozoa is a consequence of E. coli adhesion or some soluble factor from E. coli are involved. Highly motile preparation of spermatozoa from normozoospermic patients were obtained by ‘swim up’ procedure. Study was carried out in three parts. Motility parameters were analysed by light microscopy and Nomarski differential interference contrast microscopy directly, and 30 min, 2, 4, 6 h and overnight after inoculation. In a second series of experiments, bacterial replication was inhibited by addition of five antibiotics amoxicillin, ciprofloxacin, erythromycin, gentamicin and penicillin G. The effect of E. coli culture filtrates on sperm motility was investigated. In a third series, E. coli growth was suppressed by heating at high temperature (1000 C) for 30 min. E. coli decreased the sperm motility and viability by agglutination immediately after inoculation and effect increases as the time of incubation increases. Instead of this, both the culture medium in which E. coli growth was inhibited (antibiotic and heating), was not able to cause any change in motility and viability. Further, completion of incubation at 6 h the percentage of motile spermatozoa left is 11% that is very low as compared to control. This shows that E. coli may be carrying some adhesion sites through which it binds to human spermatozoa and causes agglutination.