This work aimed to assess the influence of removal of seminal plasma (RSP) and caffeine supplementation upon quality and fertilizing ability of Egyptian buffalo-bulls' spermatozoa post 6 days of hypothermic preservation at 5ºC. Semen samples were collected from five buffalo-bulls pooled, then separated into two main parts. First part was centrifuged (washed) twice to remove the seminal plasma, then the sperm plugs were re-suspended in camel skim milk extender to a volume equal to that of semen pre-washing. Second part was non-centrifuged (unwashed). Both washed and unwashed semen were diluted (1:4) with camel skim milk extender containing 10% egg yolk supplemented with caffeine at different concentrations (0,5,10 and 15 mM) and gradually cooled to 5°C then preserved at this temperature for 6 days. Data elaborated that RSP enhanced (P<0.01) sperm motility, head-to-head agglutinated spermatozoa and conception rate percentages, while dead, abnormalities and acrosomal damage percentages of spermatozoa , as well as, leakage of ALT, AST, hyaluronidase and ALP enzymes to extracellular fluids of spermatozoa were reduced (P<0.01) post-RSP . The best (P<0.01) semen characteristics, enzymatic activities and fertilizing efficiency of buffalo-bulls' spermatozoa were registered with washed diluted buffalo-bulls semen supplemented with 10 mM caffeine. Conclusively, removal of seminal plasma is a simple, effective, and easily practicable technique, which supplies a helpful alternative to semen preserved in liquid status, especially when administrated with 10mM caffeine for optimizing quality and fertilizing efficiency of buffalo spermatozoa that can be useful for artificial insemination in Egyptian buffaloes