This study investigated the effects of dietary supplementation of fish oil on the signals of lipid metabolism involved in hepatic cholesterol and triglyceride influx and excretion in high-fat diet (HFD)-fed rats. Fish oil (FO) repressed body (HFD, 533 +/- 18.2 g; HFD+FO, 488 +/- 28.0 g, p < 0.05) and liver weights (HFD, 5.7 +/- 0.6 g/100 g of body weight; HFD+FO, 4.8 +/- 0.4 g/100 g of body weight, p < 0.05) in HFD-fed rats. Fish oil could also improve HFD-induced imbalance of lipid metabolism in blood, liver, and adipose tissues including the significant decreases in plasma and liver total cholesterol (TC) (plasma-HFD, 113 +/- 33.6 mg/dL; HFD+FO, 50.0 +/- 5.95 mg/dL, p < 0.05; liver-HFD, 102 +/- 13.0 mg/dL; HFD+FO, 86.6 +/- 7.81 mg/dL, p < 0.05), blood, liver, and adipose triglyceride (TG) (blood-HFD, 52.5 +/- 20.4 mg/dL; HFD+FO, 29.8 +/- 4.30 mg/dL, p < 0.05; liver-HFD, 56.2 +/- 10.0 mg/dL; HFD+FO, 30.3 +/- 5.28 mg/dL, p < 0.05; adipose-HFD, 614 +/- 73.2 mg/dL; HFD+FO, 409 +/- 334 mg/dL, p < 0.05), and low density (HFD, 79.8 +/- 40.9 mg/dL; HFD+FO, 16.6 +/- 5.47 mg/dL, p < 0.05) and very-low-density (HFD, 49.7 +/- 33.3 mg/dL; HFD+FO, 10.4 +/- 3.45 mg/dL, p < 0.05) lipoprotein and the significant increases in fecal TC (HFD, 12.2 +/- 0.67 mg/dL; HFD+FO, 16.3 +/- 2.04 mg/dL, p < 0.05) and TG (HFD, 2.09 +/- 0.10 mg/dL; HFD+FO, 2.38 +/- 0.22 mg/dL, p < 0.05) and lipoprotein lipase activity of adipose tissues (HFD, 16.6 +/- 3.64 mu M p-nitrophenol; HFD+FO, 24.5 +/- 4.19 mu M p-nitrophenol, p < 0.05). Moreover, fish oil significantly activated the protein expressions of hepatic lipid metabolism regulators (AMPK alpha and PPAR alpha) and significantly regulated the lipid-transport-related signaling molecules (ApoE, MTTP, ApoB, Angptl4, ApoCIII, ACOX1, and SREBPF1) in blood or liver of HFD-fed rats. These results suggest that fish oil supplementation improves HFD-induced imbalance of lipid homeostasis in blood, liver, and adipose tissues in rats.