Using primary cultured piglet sertoli cells as cell model, different concentration of gossypol(0, 2.5, 5, 10, 20, 40, 80, 160 μmol/L) or 40 μmol/L gossypol plus 800 μmol/L acetylcysteine(NAC) for 24 h were applied in the study. The cell proliferation, intracellular level of reactive oxygen species(ROS), malondialdehyde(MDA) and supper oxide dismutase(SOD) activity, lactate dehydrogenase(LDH) activity in culture medium and cell apoptosis were measured. The results showed that the cell proliferation of piglet sertoli cells was significantly inhibited by gossypol in a concentration- dependent manner. With the concentration of gossypol no less than 10 μmol/L, the cell proliferation rate was extremely significantly decreased; gossypol significantly increased intracellular ROS levels, significantly decreased SOD activity, increased intracellular MDA content and LDH activity in culture medium; NAC co-treatment significantly inhibited gossypol-induced increased intracellular ROS levels, inhibited cell proliferation and increased apoptosis. These results indicated that gossypol could induce piglet sertoli cell apoptosis, which was associated with excess of ROS-induced oxidative stress.