There is increasing evidence that 17β-estradiol (E₂) directly influences the quality of maturing oocytes and thus the outcome of assisted reproduction treatment. Although Cordyceps sinensis (CS) mycelium, a Chinese herbal medicine, is believed to enhance libido and fertility in both sexes, the mechanism of its effect in women has not been determined. The aim of the present study was to evaluate the effects of CS on steroidogenic enzyme expression and E₂ biosynthesis in human granulosa-lutein cells (GLC). We found that CS induced E₂ production by GLC in a dose- and time-dependent manner and that a 3-h treatment with CS induced increased levels of mRNAs coding for the P450 side chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase. Western blot analysis demonstrated that, after treatment with CS for 3 h, protein levels of steroidogenic acute regulatory protein (StAR) and aromatase were upregulated while P450scc and 3β-HSD levels showed no substantial change. New protein synthesis was required for CS-induced E₂ production because it was abrogated by cycloheximide pretreatment. Addition of 22(R)-hydroxycholesterol, thus bypassing the need for StAR protein, did not induce as much E₂ production as CS treatment, indicating that upregulation of StAR protein was not the only factor contributing to CS-induced steroidogenesis. Cotreatment of GLCs with CS and aminoglutethimide, an aromatase inhibitor, completely abolished CS-induced E₂ production. In conclusion, treatment of GLCs with CS results in increased E₂ production due, at least in part, to increased StAR and aromatase expression. These data may help in the development of treatment regimens to improve the success rate of in vitro fertilization.