The mechanism underlying the efficacy of different bile acids (BAs) as feed additives to prevent or treat fatty liver disease in cultured fish remains poorly understood. This study compared the effect of different types of BAs on farnesoid X receptor (FXR) activity and lipid accumulation in grass carp. A survey revealed that grass carp (608.24 ± 17.511 g) with severe fatty liver had impaired FXR activity and increased liver total bile acids (TBA) content. In an in vitro study, four typical BAs, chenodeoxycholic acid (CDCA), cholic acid (CA), lithocholic acid (LCA), and deoxycholic acid (DCA), were found to significantly upregulate the expression of FXR and small heterodimer partner (SHP) and decreased lipid droplet accumulation in the hepatocytes of grass carp. Among these, DCA and CDCA exerted the most significant effects. In an in vivo study, grass carp (6.29 ± 0.05 g) were fed 100 mg/kg CDCA, CA, LCA, and DCA for 8 weeks; DCA alone significantly reduced lipid content in the liver and total cholesterol (T-CHO), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDLC) content in the serum. In addition, DCA improved the lipid catabolic activity, inhibited the lipid anabolic activity, and alleviated inflammation in the liver. In contrast, LCA, CDCA, and CA had the opposite effect on these indices. Mechanistically, DCA had a dominant impact on activating FXR in the liver, whereas the other BAs either inhibited gut FXR activity or had the risk in damaging liver cells. In conclusion, our results suggest that different BAs have varying effects on regulating fat accumulation, and DCA appears to be a particularly effective BA in suppressing fat accumulation via FXR pathway activation in the liver of grass crap.