This study aimed to evaluate the effect of Sephadex column filtration technique of post-diluted semen on freezing ability and fertilizing capacity of buffalo semen. Four Egyptian buffalo bulls were used for semen collection. Semen was extended (37^oC) at a rate of 1:20 and filtered using Sephadex column with G-75 (T2) or G-100 (T3) as compared to un-filtrated semen (T1). Semen in all treatments was evaluated for progressive motility, livability, abnormality and intact acrosome spermatozoa in post-diluted, post-equilibrated and post-thawing semen. Pregnancy diagnosis was performed at 50 d post-insemination. Results showed that T2 and T3 improved (P<0.05) percentage of motility, livability, abnormality and intact acrosome spermatozoa in post-diluted, post-equilibrated and post-thawed semen as compared to T1, being better with T2 (G-75) than T3 (G-100). For buffalo cows inseminated with post-thawed semen, conception rate was 73.3, 63.3 and 56.7% in T2, T3 and T1, respectively (P<0.05).     The present results indicate that using Sephadex (G-75) column filter technique has beneficial effects on improving semen quality and fertilizing capacity of buffalo spermatozoa by removal the abnormal and dead spermatozoa in buffalo semen, which can have an adverse effect on their fertilizing potential..