Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. It encompasses a broad spectrum of liver conditions ranging from simple steatosis to the more severe and progressive disease, non-alcoholic steatohepatitis (NASH) that can lead to hepatocellular carcinoma (HCC). Obesity and related metabolic syndrome and insulin resistance are important risk factors for the development of NAFLD, NASH and HCC. We investigated the impact of DUSP3 deficiency in metabolic syndrome manifestations and in HCC using a knockout (KO) mouse model. While aging, DUSP3-KO mice became obese and exhibited insulin resistance. These phenotypes were exacerbated under HFD and were accompanied by NAFLD and associated liver damages. In addition, DEN administration combined to HFD led to a rapid HCC development compared to WT mice. DUSP3-KO mice had more serum triglycerides, cholesterol, AST and ALT than control WT mice under both regular chow diet (CD) and HFD. The level of fasting insulin was higher compared to WT mice, though, fasting glucose as well as glucose tolerance were similar to those of control mice. At the molecular levels, HFD led to a decrease of DUSP3 expression at both protein and RNA levels. Under CD, DUSP3 deletion was associated with increased phosphorylation of the insulin receptor (IR) and subsequently, with higher activation of the downstream signaling pathway. The difference of IR phosphorylation was maintained between mutant and WT mice under HFD. However, IR downstream signaling pathway was equally activated in both groups of mice. Liver RNA sequencing and differential expression genes analysis demonstrated the effect of the diet on the expression of several genes, including genes from lipid metabolism. Gene set enrichment analysis highlighted pathways specifically regulated in DUSP3-KO mice, such as fatty acid metabolism and DNA repair pathways. Collectively, our results support a new role for DUSP3 in obesity, insulin resistance, NAFLD and liver damage.