The objectives of this study were to investigate the effects of basal media, supplements, including mixture of 5 mg/ml insulin, 5 mg/ml transferrin and 5 g/ml selenium (ITS), bovine serum albumin (BSA), sodium pyruvate (PNa) and epidermal growth factor (EGF) compared with serum on the proliferation of diethylstilbestrol-treated C57BL/6J mouse primary granulosa cells (GCs) to develop a serum-free culture system and to use this system to evaluate the effects of these supplements on GCs processes of steroidogenesis and related regulation mechanism. DMEM/F12, McCoys 5a, RPMI Medium 1640, Medium 199 and a-MEM were chosen as basal media, and McCoys 5a proliferated the GCs greatly (p < 0.05) with a better attachment and morphology. Granulosa cells showed a better proliferation status in serum-containing media than serum-free media, BSA, ITS, PNa and EGF increased the proliferation of GCs in serum-free system gradually, while the latter three had the same effect on supporting GCs proliferation with serum. After 2-day culture, the cells cultured remained P450arom positive, which sustained a normal morphology and there was no sign of apoptosis when supplements were added in the serum-free condition. Only ITS induced oestradiol production, while ITS, BSA and PNa enhanced progesterone secretion significantly indicating the promotion of differentiation. And these supplements functioned partly through increasing the transcription level of StAR, 3 beta-HSD, P450scc or P450arom. In conclusion, McCoys 5a medium was a better basal medium, and ITS and PNa promoted both the proliferation and differentiation of primary GCs in serum-free culture system, showing a satisfactory state for GCs physiological study.