The mouse is an established and popular animal model for studying reproductive biology. Epididymal mouse sperm, which lack exposure to secretions of male accessory glands and do not precisely represent ejaculated sperm for the study of sperm functions, have been almost exclusively used in studies. We compared ejaculated and epididymal sperm in an in vitro fertilization setting to examine whether ejaculated sperm enter cumulus-oocyte complexes more efficiently. In order to prepare sperm for fertilization, they were incubated under capacitating conditions. At the outset of incubation, ejaculated sperm stuck to the glass surfaces of slides and the incidences of sticking decreased with time; whereas, very few epididymal sperm stuck to glass at any time point, indicating differences in surface charge. At the end of the capacitating incubation, when sperm were added to cumulus-oocyte complexes, the form of flagellar movement differed dramatically; specifically, ejaculated sperm predominantly exhibited increased bending on one side of the flagellum (a process termed pro-hook hyperactivation), while epididymal sperm equally exhibited increased bending on one or the other side of the flagellum (pro-hook or anti-hook hyperactivation). This indicates that accessory sex gland secretions might have modified Ca2+ signaling activities in sperm, because the two forms of hyperactivation are reported to be triggered by different Ca2+ signaling patterns. Lastly, over time, more ejaculated than epididymal sperm entered the cumulus oocyte complexes. We concluded that modification of sperm by male accessory gland secretions affects the behavior of ejaculated sperm, possibly providing them with an advantage over epididymal sperm for reaching the eggs in vivo.