Abstract: Bacillus subtilis is widely used in aquaculture as a probiotic. However, few studies have been conducted to examine the efect of B. subtilis on liver lipid metabolism. A total of 135 healthy grass carp (50.24 ± 1.38 g) were randomly divided into three groups: control (Con), high-fat diet (HF), and high-fat diet + B. subtilis (HF + B. subtilis), and fed for 8 weeks. The results showed that compared with the HF group, the weight gain rate (WGR) signifcantly increased (P < 0.05) and the hepatic lipid content, serum low-density lipoprotein cholesterol (LDL-C), and aspartate aminotransferase (AST) decreased in the group supplemented with B. subtilis (P < 0.05). Moreover, the hepatic mRNA expression of fatty acid synthase (FAS) was signifcantly down-regulated and the carnitine palmitoyl transferases (CPT1α1a) were up-regulated in the HF + B. subtilis group compared to the HF group (P < 0.05), respectively. Additionally, in the HF + B. subtilis group, glutathione (GSH) signifcantly increased (P < 0.05), while hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents signifcantly decreased compared to the HF group (P < 0.05). B. subtilis may reduce the hepatic lipid content by inhibiting its synthesis and promoting β-oxidation of fatty acids. B. subtilis may also alleviate dyslipidaemia and prevent oxidative damage in the liver caused by the high-fat diet of grass carp. Hence, dietary supplementation with B. subtilis shows promise as a therapeutic or preventive tool against fatty liver disease.