This study was designed to determine possible effects of human kisspeptin-10 (KP-10) on motility spectrum of buffalo bull spermatozoa. Semen was collected from three adult Nili-Ravi buffalo bulls twice a week for three-weeks, split into five aliquots and diluted with different doses (0, 0.2, 2.0, 20.0 and 40.0 µM) of KP-10 in 0.1% BSA-PBS. The aliquots were incubated for 2, 6, 12 and 24 min at 37°C and evaluated through Computer-Assisted Sperm Analysis (CASA) system for sperm motility parameters. Ethanol fixed smears were processed for immuno-cytochemical detection of KP. Results showed that KP-10 exposure (mostly at 20.0 and 40.0 µM) for different incubation time-points significantly (P<0.05) improved CASA sperm motilities, velocities, and rapid subpopulation than control. Furthermore, buffalo bull spermatozoa showed KP-10 expression in their tail region. This study identified the stimulatory effect of human KP-10 on buffalo bull sperm motility parameters during in vitro incubation. Thus, human KP-10 can be potentially used to enhance quality of buffalo bull sperm during incubation at 37°C in vitro.