BACKGROUND AND AIMS NAFLD has become the most common liver disease worldwide but lacks a well-established pharmacological therapy. Here, we aimed to investigate the role of an E3 ligase SH3 domain-containing ring finger 2 (SH3RF2) in NAFLD and to further explore the underlying mechanisms. METHODS AND RESULTS In this study, we found that SH3RF2 was suppressed in the setting of NAFLD across mice, monkeys, and clinical individuals. Based on a genetic interruption model, we further demonstrated that hepatocyte SH3RF2 deficiency markedly deteriorates lipid accumulation in cultured hepatocytes and diet-induced NAFLD mice. Mechanistically, SH3RF2 directly binds to ATP citrate lyase, the primary enzyme promoting cytosolic acetyl-coenzyme A production, and promotes its K48-linked ubiquitination-dependent degradation. Consistently, acetyl-coenzyme A was significantly accumulated in Sh3rf2-knockout hepatocytes and livers compared with wild-type controls, leading to enhanced de novo lipogenesis, cholesterol production, and resultant lipid deposition. CONCLUSION SH3RF2 depletion in hepatocytes is a critical aggravator for NAFLD progression and therefore represents a promising therapeutic target for related liver diseases.