Earlier in our laboratory, sperm immobilization factor (SIF) isolated from Escherichia coli has shown admirable contraceptive efficacy in female mice and thus generated interest in evaluating the same as a potential candidate for male contraception. Keeping this in mind, the present study was aimed at exploitation of SIF as contraceptive agent in male mouse model after intra testis administration. For this, Balb/c mice were administered with different concentrations (10, 50, 100, 200, or 400µg) of SIF and sacrificed on days 3,7,14,21,30,45,60, and 90. After sacrifice, results in terms of change in body weight, tissue somatic indices, seminal parameters, histology, hematological parameters, lipid peroxidation, testosterone level, and cytokines level were evaluated. The weight profile and TSI of all experimental groups showed no significant change. In case of seminal parameters, 400μg of SIF led to azoospermia up to 90 days and tissue histology also revealed hypo-spermatogenesis. No significant change was observed in haematological parameters after SIF (400µg) administration however, a significant decrease was observed in testosterone levels. The oxidative status and cytokines profile was also affected after SIF (400µg) inoculation. Thus, when administered via intra testis route, SIF can lead to azoospermia and poses to be a potent and safe antifertility agent.