Seminal fluid inhibits sperm capacitation mainly because of its high cholesterol content. Prostasomes are the main source of cholesterol in seminal fluid. They are known to have numerous protective properties and are able to transfer proteins and lipids to spermatozoa, but their impact on capacitation and acrosome reaction (AR) is not yet well understood. The aim of this study was to determine the effects of prostasomes on human sperm capacitation and AR. After 80% Percoll selection, freshly ejaculated human spermatozoa were incubated for 3 h under capacitating conditions with prostasomes, phosphodiesterase inhibitor 3-iso-butyl-methylxantine (IBMX), or a combination of prostasomes and IBMX. Physiological concentration of prostasomes significantly decreased tyrosine phosphorylation levels of human sperm capacitation markers P110 and P80 (p < 0.01), and the proportions of capacitated (p < 0.05) and acrosome-reacted spermatozoa (p < 0.05). Prostasomes significantly increased the proportion of spermatozoa that did not incorporate propidium iodide and significantly attenuated the effect of IBMX on P110 tyrosine phosphorylation. Prostasomes had no effect on the pHi increase associated with capacitation. They significantly increased intracellular cAMP concentration ([cAMP]i) and, when prostasomes and IBMX were present together, [cAMP]i was further increased. To our knowledge, this is the first study to show clearly that prostasomes inhibit capacitation and spontaneous AR.