Simple Summary Hepatic HNF4 alpha inhibition can attenuate bile acid toxicity in NAFLD. However, it can aggravate hepatic steatosis, so it is necessary to study the optimal timing of HNF4 alpha inhibition and the development of surrogate markers that can predict it. It is known that bile acid-induced toxicity, along with lipotoxicity, plays a very important role in NAFLD patients. Except in very exceptional conditions, lipotoxicity caused by free fatty acids and bile acid toxicity caused by bile acid always coexist in the NAFLD human condition. In this respect, we believe that inhibiting HNF4 alpha is more effective as a therapeutic strategy for NAFLD. Hepatocyte nuclear factor 4 alpha (HNF4 alpha) is a key master transcriptional factor for hepatic fat and bile acid metabolic pathways. We aimed to investigate the role of HNF4 alpha in non-alcoholic fatty liver disease (NAFLD). The role of HNF4 alpha was evaluated in free fatty acid-induced lipotoxicity and chenodeoxycholic acid (CDCA)-induced bile acid toxicity. Furthermore, the role of HNF4 alpha was evaluated in a methionine choline deficiency (MCD)-diet-induced NAFLD model. The overexpression of HNF4 alpha reduced intracellular lipid contents and attenuated palmitic acid (PA)-induced lipotoxicity. However, the protective effects of HNF4 alpha were reversed when CDCA was used in a co-treatment with PA. HNF4 alpha knockdown recovered cell death from bile acid toxicity. The inhibition of HNF4 alpha decreased intrahepatic inflammation and the NAFLD activity score in the MCD model. Hepatic HNF4 alpha inhibition can attenuate bile acid toxicity and be more effective as a therapeutic strategy in NAFLD patients; however, it is necessary to study the optimal timing of HNF4 alpha inhibition.