Vitamin D Receptor (VDR) is expressed in both animal and human ovarian tissue, however, the role of vitamin D in human ovarian steroidogenesis is unknown. Cultured human ovarian cells were incubated in tissue culture medium supplemented with appropriate substrates, with or without 50 pM-150 pM or 50 nM-150 nM of 1,25-(OH)2D3, and in the presence or absence of insulin. Progesterone, testosterone, estrone, estradiol, and IGFBP-1 concentrations in conditioned tissue culture medium were measured. Vitamin D receptor was present in human ovarian cells. 1,25-(OH)2D3 stimulated progesterone production by 13% (p<0.001), estradiol production by 9% (p<0.02), and estrone production by 21% (p<0.002). Insulin and 1,25-(OH)2D3 acted synergistically to increase estradiol production by 60% (p<0.005). 1,25-(OH)2D3 alone stimulated IGFBP-1 production by 24% (p<0.001), however, in the presence of insulin, 1,25-(OH)2D3 enhanced insulin-induced inhibition of IGFBP-1 production by 13% (p<0.009). Vitamin D stimulates ovarian steroidogenesis and IGFBP-1 production in human ovarian cells likely acting via vitamin D receptor. Insulin and vitamin D synergistically stimulate estradiol production. Vitamin D also enhances inhibitory effect of insulin on IGFBP-1 production.