Objective To explore the mechanism of Canopy homolog 2 (CNPY2) and aerobic exercise through nuclear factor kappa-B (NF-κB) signal pathway in non-alcoholic fatty liver disease (NAFLD) induced by high fat diet. Methods Male CNPY2 knockout (CNPY2 KO) mice and wild (WT) mice aged (12±1) weeks were randomly divided into control group (CON),hyperlipidemic model group (MOD), and hyperlipidemic model exercise group (MOD+EX) after one week of adaptive feeding.CON group was fed with normal diet, and MOD group and MOD+EX group were fed with high fat diet until the end of 18 weeks.From the 10^th week, the mice in the MOD+EX group received adaptive treadmill training for a week, followed by continuous exercise intervention until the end of the 18-week experiment. The serum levels of TC, TG, LDL-C, HDL-C, ALT, and AST were detected by automatic biochemical analyzer, the pathological morphology of liver was analyzed by HE staining and oil red O staining, the protein expressions of CNPY2, IκB α, p-IκBα, NF-κB, and NF-κB in liver were detected by Western Blot, thelevels of TNF-α and IL6 in liver were detected by ELISA, and the expressions of NF- κB mRNA, TNF- α mRNA, and IL-6 mRNA in liver were detected by q RT-PCR. The data were analyzed by single factor analysis of variance and independent sample t-test. Results The expression of CNPY2 in MOD group was higher than that in CON group (t=-5.730, P=0.001) while the expression of CNPY2 in MOD+EX group was lower than that in MOD group (t=3.714, P=0.010).Compared with CON group, the serum levels of TC, TG, LDL-c, ALT, and AST in MOD group of WT mice and CNPY2 KO mice were higher (WT: t=-13.325, P < 0.001; t=-4.889, P < 0.001; t=-10.442, P < 0.001; t=-3.500, P=0.003; t=-15.122, P <0.001; CNPY2 KO: t=-6.910, P < 0.001; t=-4.962, P < 0.001; t=-7.457, P < 0.001; t=-4.584, P < 0.001; t=-7.336, P <0.001).The levels of TNF-α, IL-6, p-IκBα/IκBα, p-NF-κB/NF-κB, NF-κB mRNA, TNF-α mRNA, and IL-6 mRNA in liver were increased significantly (WT: t=-25.179, P < 0.001; t=-21.043, P < 0.001; t=-9.177, P < 0.001; t=-12.207, P < 0.001; t=-5.205, P=0.002; t=-6.910, P < 0.001; t=-4.802, P=0.003; CNPY2 KO: t=-18.878, P < 0.001; t=-21.840, P < 0.001; t=-14.033, P < 0.001; t=-12.511, P < 0.001; t=-6.870, P < 0.001; t=-9.546, P < 0.001; t=-8.303, P < 0.001), HDL-C level decreased (WT: t=11.695, P < 0.001; CNPY2 KO: t=6.598, P < 0.001), and hepatocyte steatosis with large amount of lipid droplets was observed. Compared with MOD group, the above-mentioned indexes were effectively improved in WT mice and CNPY2 KO mice in MOD+EX group. Compared with WT mice, the above-mentioned indexes of CNPY2 KO mice were improved effectively. Conclusion CNPY2 regulates NF-κB signal pathway and participates in the formation and development of NAFLD. Both CNPY2 gene deletion and aerobic exercise can improve NAFLD, which may be related to the decrease of liver CNPY2 expression, inhibition of NF-κB signal pathway, down-regulation of liver inflammatory cytokines and reduction of liver inflammation.