Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) has been used as a traditional Chinese medicine for dysfunction of the endocrine system. However, there have been few studies on the effects of adlay seed on the endocrine system. In the present study, both the in vivo and in vitro effects of methanolic extracts of adlay hull (AHM) on progesterone synthesis were studied. AHM was partitioned with four different solvents: water, 1-butanol, ethyl acetate, and n-hexane. Four fractions, namely, AHM-Wa (water fraction), AHM-Bu (1-butanol fraction), AHM-EA (ethyl acetate fraction), and AHM-Hex (n-hexane fraction), were respectively obtained. Granulosa cells (GCs) were prepared from pregnant mare serum gonadotropin-primed immature female rats and were challenged with different reagents, including human chorionic gonadotropin (hCG; 0.5 IU/ml), 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP; 0.1 mM), forskolin (10 microM), 25-OH-cholesterol (10 microM), and pregnenolone (10 microM), in the presence or absence of AHM (100 microg/ml). The functions of steroidogenic enzymes, including protein expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage enzyme (P450scc), protein kinase A (PKA), and aromatase activity, were investigated. The expression of StAR mRNA was also explored by using real-time reverse transcription-polymerase chain reaction. In the in vivo study, AHM decreased plasma progesterone and estradiol levels after an intravenous injection of AHM (2 mg/ ml/kg). In the in vitro studies, AHM decreased progesterone and estradiol via inhibition of (i) the cAMP-PKA signal transduction pathway, (ii) cAMP accumulation, (iii) P450scc and 3beta-HSD enzyme activities, (iv) PKA, P450scc and StAR protein expressions and StAR mRNA expression, and (v) aromatase activity in rat GCs. These results suggest that AHM decreased the production of progesterone via mechanisms involving the inhibition of the cAMP pathway, enzyme activities, and the protein expressions of P450scc and StAR in rat GCs.