The effect of Vitamin E on the proliferation of ovine Sertoli cells was investigated. Sertoli cells were isolated and treated with various amounts of Vitamin E (0 μM, 400 μM, 800 μM, 1000 μM, 1200 μM, 1400 μM and 1600 μM) for 24 h. We found that at the concentration of 1200 μM, Vitamin E promoted Sertoli cell proliferation very effectively. It also increased the proportion of cells in the G1 phase while reduced that in the S and G2/M phases, suggesting that its effect on Sertoli cell proliferation is achieved by enhancing progression through the cell cycle. In addition, Vitamin E significantly up-regulated the transcript level of the PDPN, BMP6, AMPKα, GSK3β, Myc, and CDK6 genes and down-regulated that of PPARγ, Cyclin B1 and CDK4 as determined by qRT-PCR. Western blot analysis revealed that the expression of BMP6 and PDPN was also upregulated at the protein level, in accordance with the results of the qRT-PCR. Taken together, Vitamin E promoted Sertoli cell proliferation by affecting the expression of genes that regulate cell division and the cell cycle; this indicates that it can have a positive effect on sheep reproductive performance.