Abstract: OBJECTIVE   To study the effect of obacunone (OBA) on the synthesis and secretion of adrenal cortex hormones.METHODS   Y1 mouse adrenocortical tumor cells were cultured in vitro, and 2.5-160 μmol/L OBA was intervened with for 24 h. MTT assay was used to detect cell viability. Cells were treated with 80 μmol/L OBA for 24 h, flow cytometry was used to detect cell cycle, and confocal microscope was used to detect cell mitochondrial membrane changes. The content of corticosterone in cell secretion fluid was detected by ELISA. Cells were intervened with 40-160 μmol/L OBA for 24 h, and 80 μmol/L OBA for 24-48 h. The qPCR was used to detect corticosterone synthase mRNA expression, and Western blot was used to detect its protein expression.RESULTS   Compared with the control group, 2.5-40 μmol/L OBA inhibited cell growth with a rate of about 35% (P < 0.05), and 160 μmol/L OBA inhibited cell growth with a rate of more than 60% (P < 0.01). OBA showed a significant increase in G1 cells and promoted the enhancement of mitochondrial membrane brightness (P < 0.05), while significantly inhibited the expression of Mfn1 and Mfn2 proteins in mitochondrial membranes and the corticosterone secretion (P < 0.05). Furthermore, it was found that 40-160 μmol/L OBA significantly inhibited the expressions of Cyp11a1, Cyp11b1, Hsd3b2, SF-1 genes (P < 0.01), and 160 μmol/L OBA significantly enhanced the expressions of Star, Cyp21a1, Nr4a1, Nr4a2 genes (P < 0.01). 80 μmol/L OBA within 48 h of continuous administration, significantly inhibited the expressions of Cyp11a1, Cyp21a1, Cyp11b1, and Hsd3b2 genes (P < 0.01), and StAR and CYP11A1 protein, but promoted the expression of Star gene (P < 0.01).CONCLUSION   OBA can inhibit the corticosterone synthesis by corticosterone in adrenal cortex cells, which may be related to cell cycle arrest and the expression of steroid synthase on mitochondrial membrane.