The present was conducted to separate heparin binding proteins (HBP) of capacitated sperm extracts and determine their relationship with acrosome reaction and semen fertility of bulls (n=30). Fourteen immunoreactive bands in capacitated spermatozoa of bulls (135, 100, 75, 70, 65, 55, 48, 45, 33, 31, 26, 24, 20 and 18 kDa) were detected in western blots following incubation with anti-HBP antibody. The frozen-thawed semen was evaluated for first service conception rate (FSCR) and percent acrosome reaction and linked to HBP. In capacitated spermatozoa, bulls positive for 70, 31, 20 and 18 kDa had significantly higher (P < 0.05) FSCR (42.3 ± 5.1 vs 32.9 ± 3.9%, 45.0 ± 6.5 vs 34.1 ± 3.5%, 60.0 ± 10.0 vs 35.2 ± 3.3% and 42.9 ± 5.1 vs 31.9 ± 3.7%), respectively, as compared to their negative counterparts. Although non-significant, FSCR was reasonably higher (P > 0.05) in bulls positive for 135 and 100 kDa HBP than in their negative contemporary mates and exhibited an increase of 7.4% for the two proteins. A significantly higher (P < 0.05) rate of acrosome reaction was recorded in bulls with HBP of 135, 100, 70 and 18 kDa in comparison to their contemporary mates with a difference of 7.4, 7.4, 8.7 and 9.4%, respectively. Alternatively, FSCR and acrosome reaction of bulls with 75, 55 and 48 kDa were lower (P < 0.05) than their corresponding counterparts. In conclusion, HBP of 135, 100, 70 and 18 kDa did seem to activate in vitro sperm acrosome reaction and subsequent fertility of bulls.