Insulin resistance is a major feature and pathogenic factor of nonalcoholic fatty liver disease (NAFLD). Theoretically, genetic variation in candidate genes related to insulin resistance may contribute to the pathogenesis of NAFLD. The purpose of this study was to identify potentially pathogenic genes involved in NAFLD and insulin resistance that have not yet been discovered. This study yielded five important discoveries. 1. A total of 21 co-differentially expressed genes in both the NAFLD and insulin-resistance groups were identified from the pool containing thousands of genes via the significance analysis of microarrays method. 2. MAP kinase-interacting serine/threonine kinase 2 (Mknk2) was the unique gene to be identified that is involved in the insulin signaling pathway and Mitogen Activated Protein Kinase signaling pathway according to the Kyoto Encyclopedia of Genes and Genomes database. 3. Mknk2 mRNA and protein expression were dose-dependently up-regulated by palmitic acid (PA) in mouse primary hepatocytes. 4. Western blotting analysis and quantitative real-time PCR confirmed that Mknk2 affected the expression of acetyl-CoA carboxylases-1 and fatty acid synthase. 5. The inhibition of Mknk2 alleviated PA-induced insulin resistance, whereas the overexpression of Mknk2 resulted in the aggravation of insulin resistance in PA-treated hepatocytes. Therefore, we predict that MKNK2 may be a key protein related to NAFLD and insulin resistance.