Objective To explore the effects of TNF-? on the function of Leydig cells, in order to elucidate the possible mechanisms involved. Methods Highly pure primary Leydig cells were obtained by Percoll discontinuous density gradient method. HE stain was used to observe the morphology of the cultured cells. The Leydig cells were treated with different doses of rat TNF-? (0.1, 1, 10, 100 ng/ml) for 48h, and then the supernatants of culture medium were collected every 24 hours at the 1st, 2nd, 3rd and 4th day. The testosterone level in the supernatant was measured by radioimmunoassay. The proliferation and TNF-?-induced apoptosis of Leydig cells were examined by MTT assay, flow cytometry (FCM) as well as acridine orange (AO) stain. Results The purity of Leydig cells was 70%-80% after purification with Percoll discontinuous density gradient method. The Leydig cells were rich in cytoplasm, which contained some secretory granules and round nucleus. After TNF-? treatment in different concentrations (0.1, 1, 10, 100ng/ml) for 24h, the inhibition rates of TNF-? on testosterone secretion of Leydig cells were 22.0%, 35.0%, 53.0% and 74.8%, respectively, and the decrease showed a time-dependent manner, but no statistically significant difference was found in each group at every time point except for 100ng/ml group. High concentration of TNF-? (10 and 100 ng/ml) could inhibit the proliferation and promote apoptosis of Laydig cells. Compared to control group, the inhibition rate and apoptosis rate in 10ng/ml and 100ng/ml group showed significant difference (respectively 38.4%?4.1%, 76.4%?8.7% and 13.2%?1.1%, 26.4%?5.8%). In addition, significant apoptosis could be seen in the high concentration groups as shown with AO staining. Conclusion The present study suggests that TNF-? can inhibit the basal testosterone secretion of Leydig cells, which might be related to the inhibition and apopotosis-induced effects of high concentration of TNF-?.