Icariin (ICA), a major constituent of flavonoids from the Chinese medical herb Epimedium brevicornum Maxim, is found to be protective for male reproductive ability, with the underlying mechanism largely unknown. Our study here investigated the effects of ICA on Sertoli cells, which act as nurse cells for the germ cells developing. Icariin was found to stimulate Sertoli cell proliferation in a dose-dependent manner. Further study revealed that Icariin induced an obvious phosphorylation of ERK in Sertoli cells. Inhibition of activation of ERK by the ERK inhibitor U0126 nearly blocked the Icariin-induced proliferation of Sertoli cells. Taken together, our results suggest that Icariin promotes the proliferation of Sertoli cells in vitro by activating the ERK1/2 signal pathway, which might at least partially, explain the protective role of Icariin on male reproductive ability.