Reduced vitamin C (AA) is believed to be important for hormone synthesis, but its role in generating placental steroids needed to maintain pregnancy and fetal development is not clear. It is also not known whether AA enters placental cells via sodium-dependent vitamin C transporters (SVCTs) or whether the oxidized form, dehydroascorbic acid, is taken up by glucose transporters. To determine the steroidogenic effect of AA and the role of SVCT2 in AA-induced steroidogenesis, we tested the effects of AA treatment and SVCT2 knockdown on steroidogenesis in human choriocarcinoma cell lines. AA treatment of JEG-3, BeWo, and JAR cells for 48 h dose-dependently increased progesterone and 17-estradiol levels. In JEG-3 cells, AA increased the mRNA expression of P450 cholesterol side-chain cleavage enzyme (P450scc), 3-hydroxysteroid dehydrogenase type 1 (3-HSD1), and aromatase, key enzymes for steroidogenesis. Stable knockdown of SVCT2 in JEG-3 cells by retrovirally mediated RNA interference decreased the Vmax of AA uptake by approximately 50%, but Km values were not affected. SVCT2 knockdown in JEG-3 cells significantly suppressed the AA-induced mRNA expression of placental P450scc, 3-HSD1, and aromatase. This suppression of the AA-induced mRNA expression of steroidogenic enzymes subsequently decreased progesterone and 17-estradiol production. In addition, inhibition of MAPK kinase (MEK)-ERK signaling, which is a major pathway for AA-regulated gene expression, failed to affect AA-induced steroidogenesis. Our observations indicate that SVCT2-mediated AA uptake into cells is necessary for AA-induced steroidogenesis in human choriocarcinoma cell, but MEK-ERK signaling is not involved in AA-induced steroidogenesis.