Gender research shows excellent significance in the breeding and production of poultry, achieving the actual needs of poultry through targeted gender regulation could increase economic benefits. However, the mechanism of sex determination in poultry remains unclear. We respectively constructed HMGCS1 overexpression and interference vectors for lentivirus packaging. Vascular microinjection was performed when fertilized eggs developed to 2.5 days. qRT-PCR and ELISA results indicated that HMGCS1 could promote the expression of male germ-cell development-related genes (SOX9) and inhibit the expression of female germ-cell development-related genes (CYP19A1 and DMRT1). Furthermore, the synthesis of cholesterol and testosterone in the gonads were increased. In addition, the injection of lovastatin into chicken embryos, an inhibitor of cholesterol synthesis, showed a significant decrease in gonadal testosterone. This experiment revealed that HMGCS1 could regulate the synthesis of testosterone by affecting the content of cholesterol in gonads, and promote the differentiation of chicken gonads into males.