Objective:To examine adding polymyxin B (PMB) if is effective and appropriate to bull xetender, and decrease bacteria damage.Methods: PMB were added to extender (Andromed?) in different values (0, 50, 100, 500, 1000 μg/mL). The prepared samples were cryopreserved, thawed and analyzed for viability (by eosin-nigrosin staining) and plasma membrane function (by hypo osmotic solution test) in 3 different times (immediately, 1 h & 2 h later).Results: The result showed that both parameters significantly increased in 50 and 100 μgr/ML treatments. Moreover, by passing time, plasma membrane function significantly increased. But, in high dosage (1000 μg/mL), the toxicity effect of PMB was seen, and in both parameters the differences were significantly lower than the control treatment (0 μg/mL).Conclusion: Our study indicates that adding PMB to bull extender is efficient and appropriate, but, it must be mentioned that addictive amount will be due to extender composition, existence of other antibiotics and, importantly, the load of bacterial contamination in the semen.