Hypercholesterolemia is one of the major risk factors associated with the emergence and development of cardiovascular diseases (CVD) and atherosclerosis. The hypocholesterolemic effects of probiotics have been indicated by numerous studies. The 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and cytochrome P450 7A1 or cholesterol 7 alpha-hydroxylase (CYP7A1) are two important genes in choletsterol metabolism. In this study, the effects of Bacillus subtilis JQ61816 on lipid panel, hepatic enzymes and expression levels of HMGCR and CYP7A1 were investigated. Twenty-one male Wistar rats were randomly allotted to 3 experimental groups; a) negative control group (ND) fed with normal diet, b) high-fat diet group (HFD) fed with high cholesterol diet, and c) probiotic group (BS) fed with high cholesterol diet supplemented with probiotic B. subtilis. Serum analysis of treatment groups was performed to measure fasting blood sugar (FBS), lipid profile parameters, hepatic enzymes, urea, and uric acid. Our results showed that B. subtilis could reduce the level of total cholesterol, triglycerides, and LDL and it also could increase high-density lipoprotein (HDL) level. Moreover, alanine transaminase (ALT), aspartate transaminase (AST), and uric acid were significantly lower in BS group compare to HFD group. Furthermore, up-regulation of HMGCR and down-regulation of CYP7A1 were observed in BS group. The results of our study suggest that consumption of probiotic B. subtilis JQ61816 may prevent or decline the development of hypercholesterolemia and other cardiovascular diseases.