Objective:To evaluate the effect of adding different values of polymyxin B (PMB) to bull semen on various motility parameters of post-thawed semen such as total motility, progressive motility and velocity parameters using kinetic parameters of sperm by Computer Assisted Sperm Analysis.Methods: Gram negative bacteria release lipopolysaccharide, which induces the apoptotic pathway. Antibiotics are added to semen in order to prevent bacterial contaminations in bovine semen. These antibiotics kill the bacteria especially gram negative bacteria. Therefore, their endotoxins are released during bacteriolysis and bind to the head region and midpiece of sperm. PMB is a bactericidal antibiotic against multidrug resistant gram-negative bacteria and is able to neutralize the toxic effects of the released endotoxin. This study was performed on 3-year old Taleshi bulls. Results:The results showed both positive and negative significant effects of PMB on semen quality. Total motility and progressive motility were significantly increased (P<0.0001) by 100 μg per mL of PMB (55.2％ and 48.8％ respectively) against the control groups (43.5％ and 37.7％, respectively). Moreover, they were significantly decreased (P<0.0001) by 1000 μg per mL of PMB (35.2％ and 28.8％ respectively) against the control groups (43.5％ and 37.7％ respectively) in above-mentioned parameters. In Computer Assisted Semen Analyzer, parameter VAP was significantly decreased (P<0.04) in 1000 μg (69.6 μm/s) against the control group (78.7 μm/s). Finally, using PMB in processing cryopreserved bull semen is advised, but before using it, the rate of endotoxins must be measured.Conclusions: We advise using PMB after measuring endotoxin concentration;In vitro,in vivoand in field fertilization, adding other sperm evaluation factors such as acrosomal integrity, DNA integrity, mitochondrial function to PMB treated semen.