Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in hepatocytes. The role of SENP3 in lipid metabolism, particularly NAFLD, is unclear. Our results showed that hepatic SENP3 was up-regulated in NAFLD patients and an animal model in vivo and after loading hepatocytes with free fatty acids (FFA) in vitro. Intracellular lipid accumulation was determined in SENP3 silenced or overexpressed hepatocytes with/without FFA in vitro. Confirming a role for SENP3, gene silencing was associated in vitro with amelioration of lipid accumulation and overexpression with enhancement of lipid accumulation. SENP3 related genes in NAFLD were determined in vitro using RNA-Seq. Eleven unique genes closely associated with lipid metabolism were generated using bioinformatics. Three selected genes (apoe, a2m and tnfrsf11b) were verified in vitro, showing apoe, a2m and tnfrsf11b were regulated by SENP3 with FFA stimulation. Intrahepatic and circulating APOE, A2M and TNFRSF11B were elevated in NAFLD compared with controls. These data demonstrate the important role of SENP3 in lipid metabolism during the development of NAFLD via downstream genes, which may be useful information in the development of NAFLD. The precise role of SENP3 in NAFLD will be investigated using liver-specific conditional knockout mice in future studies.