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AIM: To evaluate the possible role of Tribble 3 (TRB3) in a rat model of non-alcoholic fatty liver disease (NAFLD) and its signal transduction mechanism. METHODS: Thi r ty Sprague-Dawley rats were randomized into three groups: normal control group,non-alcoholic fatty liver group A (fed on a highfat diet for 8 wk) and group B (fed on a high-fat diet for 16 wk). To determine the degree of hepatic steatosis in rats of each group, livers were stained with hematoxylin and eosin, and evaluated; realtime fluorescent quantitative reverse transcriptasepolymerase chain reaction was performed to measure the expression levels of TRB3 mRNA; and West blotting analysis was done to determine the expression levels of protein kinase B (Akt) and phosphorylated protein kinase B (p-Akt-Thr308, p-Akt-Ser473). RESULTS: Hepatic steatosis was evident in both NAFLD groups: mild to moderate hepatic steatosis occurred in group A, mainly as mild steatosis. Moderate to severe hepatic steatosis occurred in group B, mainly as severe steatosis. The expression level of TRB3 mRNA in group B was significantly higher than in the control group (122.28 ± 95.37 vs 3.06 ± 2.33,P = 0.001) and group A (122.28 ± 95.37 vs 5.77 ± 4.20,P = 0.001). There was no significant difference in the expression levels of Akt (1.03 ± 0.53 vs 1.12 ± 0.77,P = 0.729) and p-Akt-Thr308 (0.82 ± 0.45 vs 0.92 ± 0.38, P = 0.592) between group A and the control group. The expression level of Akt and p-Akt-Thr308 in group B was significantly lower than in group A (Akt 0.41 ± 0.16 vs 1.12 ± 0.77, P = 0.008; p-Akt-Thr308 0.47 ± 0.19 vs 0.82 ± 0.45, P = 0.036) and the control group (Akt 0.41 ± 0.16 vs 1.03 ± 0.53, P = 0.018; p-Akt-Thr308 0.47 ± 0.19 vs 0.92 ± 0.38, P = 0.010).The expression level of p-Akt-Ser473 in group A was significantly higher than in group B (1.48 ± 0.50 vs 0.81 ± 0.39, P = 0.041) as well as the control group (1.48 ± 0.50 vs 0.45 ± 0.26, P = 0.003).CONCLUSION: TRB3 blocks insulin signaling by inhibiting Akt activation, which contributes to insulin resistance. It may be an important factor in the occurrence and development of NAFLD.