Modulating steroid hormone levels is a curative and preventive measure for Cushing's syndrome, aldosteronism, and various stress-triggered symptoms. Polyphenols have been reported to inhibit steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase. However, evidence for their inhibitory effects is fragmentary because it has been determined in studies with small groups of steroid hormones. To investigate the effects of steroids on complete steroidogenic pathways, comprehensive analysis of steroid hormones is necessary. Here we cultured forskolin-stimulated NCI-H295R, a human adrenocortical carcinoma cell line, in the presence of a polyphenol and employed GC-MS to simultaneously determine the levels of nine steroid hormones (pregnenolone, progesterone, deoxycorticosterone, aldosterone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, and estradiol) in cell culture supernatant. We found that daidzein, genistein, apigenin, hesperetin, naringenin, and eriodictyol significantly reduced deoxycorticosterone and androstenedione levels (p<0.05), suggesting inhibition of 3β-HSD by these polyphenols. Apigenin was more potent than other polyphenols in increasing the levels of pregnenolone and 17α-hydroxyprogesterone, suggesting that it inhibits cytochrome P450 (CYP) 17 and CYP21, as well as 3β-HSD. Real-time reverse transcription polymerase chain reaction showed that apigenin significantly downregulated the expression levels of 3β-HSD, CYP17, and CYP21 mRNA (p<0.05). This is the first study to demonstrate the inhibitory effects of apigenin on CYP17 and CYP21.