The aim of the study was to investigate the effects of silencing the inhibin α-subunit (INHα) gene by RNAi on the mRNA levels of steroidogenic acute regulatory protein (StAR), follistatin (FST), inhibin β-subunit (INHβB), and FSH receptor (FSHR) in cultured sheep granulosa cells (GCs). The antral follicles, 3–7 mm in diameter, were dissected from the ovaries of 15 Thin-tailed Han sheep (1-1.5 years old), and then GCs were isolated from antral follicles. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to analyze the mRNA levels of StAR, FST, INHβB, and FSHR in INHα-specific small RNA interference (siRNA) − treated GCs. Knockdown of INHα mRNA by siRNA decreased the mRNA expressions of StAR and FST by 76% and 23%, respectively (P < 0.05), and increased the mRNA expressions of FSHR and INHβB by 81% and 50% in cultured sheep GCs, respectively (P < 0.05). These results indicated that silencing INHα gene by RNAi influenced the mRNA expressions of StAR, FST, INHβB and FSHR in cultured sheep GCs.