Background: TNF-alpha has a wide range of functions in processes within the ovaries, including follicular development, ovarian hormones synthesis, ovulation, granulosa cell necroptosis, and follicular atresia. Innate immune response in the ovaries is the main source of TNF-alpha. The purpose of this study is to explore the dose- and time-dependent interactions between TNF-alpha and human granulosa cells. Methods: The follicular fluids (FF) were obtained from infertile patients undergoing IVF with PCOS (n = 20) and healthy women (n = 19). Granulosa cells were cultured and treated with varying concentration of recombinant human TNF-alpha. Cell viability was detected by CCK-8 method, the levels of TNF-alpha, estradiol, and inhibin were measured with ELISA, and cell apoptosis was detected by flow cytometry. Q-PCR and western-blot were used to evaluate the expression of TNFR1/2, P450, Inhibin alpha/beta A, and caspase 3. Results: The level of TNF-alpha increased gradually with the development of follicles and was remarkably higher in the FF of PCOS patients than in that of healthy women. Treatment with TNF-alpha as low as 0.2 ng/mL promoted the proliferation of granulosa cells, thus upregulating P450 aromatase and inhibin A. On the other hand, a high concentration of TNF-alpha (0.4, 0.8, 2, and 8 ng/mL) showed dose- and time-dependent inhibitory effects on cell viability and E2 and inhibin A secretion. However, no marked differences in the expression of the two types of TNFR were observed in TNF-alpha-treated granulosa cells. Conclusions: The data presented here verify a biphasic effect of TNF-alpha on human ovarian endocrine and follicular development.