Nonalcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver disease worldwide. Triglyceride (TG) accumulation is a key pathological feature of NAFLD. Therefore, the understanding of the molecular mechanisms involved in TG accumulation may lead to the development of potential drug targets for the treatment of NAFLD. In this study, we found that zinc finger BED-type containing 3 (ZBED3) was significantly upregulated in the livers of NAFLD patients and animal models. Here, we found that ZBED3 directly interacts with polypyrimidine bundle binding protein 1 (PTBP1) and affects its binding to sterol regulatory element binding protein 1c (SREBP1c) mRNA precursors, thereby regulating mRNA stability and alternative splicing based on co-immunoprecipitation and mass spectrometry. This result reveals the specific mechanism by which ZBED3 overexpression promotes free fatty acid-induced TG accumulation in hepatocytes.