Rosmarinic acid (RA), a natural phenolic ester, is cytoprotective for male reproduction in animal models. The present study investigated the in vitro actions of RA on human sperm functions. Human sperm were exposed to 1, 10, 100, and 1000 μM RA in vitro and sperm functions were examined. The results showed that although RA did not affect human sperm viability, RA at 10-1000 μM dose-dependently reduced sperm motility, penetration ability, capacitation, and spontaneous acrosome reaction. In addition, the intracellular Ca2+ concentration ([Ca2+]i), which serve as a key regulator of sperm function, was decreased by RA (10-1000 μM) in a dose-dependent manner. Furthermore, the current of the sperm-specific potassium channel, KSPER, which is predominant for Ca2+ influx in sperm, was dose-dependently inhibited by 10-1000 μM RA. Therefore, we conclude that in vitro exposure to RA can compromise human sperm functions by decreasing sperm [Ca2+]i through the suppression of KSPER current.