Poor freezability and post-thaw sperm quality is one of the major problems in crossbred cattle limiting the success of artificial insemination and genetic improvement under field conditions. In an attempt to improve the freezability and post-thaw semen quality, the current study was undertaken to assess the effects of buffalo epididymal proteins on crossbred bull spermatozoa during cryopreservation. A preliminary study was conducted to select a suitable dose of epididymal proteins. The epididymal proteins (whole epididymal, caput, corpus and cauda epididymal proteins) were incorporated in the semen extender at 1mg/mL of the extended semen (concentration selected based on the results of preliminary study). In the control group no proteins were incorporated. A total of 36 ejaculates from six number of crossbred bulls were utilised in the study and before (pre-freeze stage) and after (after thawing) cryopreservation sperm quality was assessed. Significantly higher pre-freeze and post-thaw sperm motility, viability and acrosomal integrity was observed in whole epididymal protein added group in comparison to other epididymal protein added groups and control group. The sperm penetration distance and membrane integrity were greater in both cauda epididymal protein and whole epididymal proteins added group in comparison to rest of epididymal protein added groups and control. In pre-freeze and post-thaw stage, among the whole and cauda epididymal proteins added group revealed a significantly lower level of MDA production as compared to caput and corpus epididymal proteins added groups and control group. Collectively, the findings of the current study indicate that the buffalo epididymal proteins offer protection to cattle spermatozoa during equilibration and during ultralow temperature cryopreservation.