Twelve buffalo bulls of 3-5 years of age and 400 to 500 kg live body weights were used in the present study. The experimental work was carried out to establish the optimum condition of osmotic pressure using lactose-Na-citrate solution at different osmolarities (50, 100, 150, 200, and 300 mOsmol/L) either washed or non-washed buffalo spermatozoa. The percentage of sperm motility, grade of the progressive sperm motility (score), percentages of spermatozoa with coiled tail, and spermatozoa swelling, were determined during incubation at 37°C for 0, 5, 15, 30 and 60 minutes. The results showed that, washed buffalo bull semen increased significantly (P<0.05) the percentage of sperm motility, grade of the progressive sperm motility, percentages of spermatozoa with coiled tails and spermatozoa swelling as compared to the non washed semen. The percentage of sperm motility and grade of the progressive sperm motility were significantly (P<0.05) increased, while the percentages of spermatozoa with coiled tail and spermatozoa swelling were significantly (P<0.05) decreased in the extended buffalo bull spermatozoa either washed or non-washed with lactose-Na-citrate solution at levels of 200 or 300 as compared to 50, 100 or 150 mOsmol/L. The advancement of incubation time at 37°C for up to 60 minutes of the extended buffalo bull spermatozoa either washed or non-washed semen with lactose-Na-citrate solution at different levels of osmolarities decreased significantly (P<0.05) the percentage of sperm motility and grade of the progressive sperm motility, while increased significantly (P<0.05) the percentages of spermatozoa with coiled tail and spermatozoa swelling.