Background and Aims Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease, which has no specific pharmacological treatments partially because of the unclear pathophysiological mechanisms. Regulator of G protein signaling (RGSs) proteins are proteins that negatively regulate G protein–coupled receptor (GPCR) signaling. The members of the R4/B subfamily are the smallest RGS proteins in size, and RGS5 belongs to this family, which mediates pluripotent biological functions through canonical G protein–mediated pathways and non‐GPCR pathways. This study combined a genetically engineered rodent model and a transcriptomics‐sequencing approach to investigate the role and regulatory mechanism of RGS5 in the development of NAFLD. Approach and Results This study found that RGS5 protects against NAFLD and nonalcoholic steatohepatitis. Using RNA sequencing and an unbiased systematic investigative approach, this study found that the activation of mitogen‐activated protein kinase signaling cascades in response to metabolic challenge is negatively associated with hepatic RGS5 expression. Mechanistically, we found that the 64‐181 amino‐acid‐sequence (aa) fragment of RGS5 directly interacts with transforming growth factor beta–activated kinase 1 (TAK1) through the 1‐300aa fragment and inhibits TAK1 phosphorylation and the subsequent c‐Jun‐N‐terminal kinase (JNK)/p38 pathway activation. Conclusions In hepatocytes, RGS5 is an essential molecule that protects against the progression of NAFLD. RGS5 directly binds to TAK1, preventing its hyperphosphorylation and the activation of the downstream JNK/p38 signaling cascade. RGS5 is a promising target molecule for fine‐tuning the activity of TAK1 and for the treatment of NAFLD.