Background: Adipogenesis is an essential process in organismal development and plays a significant role in adipose tissue homeostasis. Post-transcriptional regulation of gene expression plays a key role in adipogenesis and involves many RNA-binding proteins (RBPs). In mammals, Staufen1 (STAU1) is a conserved RBP(RNA Binding Protein )consisting of several dsRBP (double strand RNA). STAU1 plays an important role in the Stau1-mediated mRNA decay (SMD) pathway, which is related to adipocyte formation, myocyte development, and neural differentiation. Klf16 (Kruppel like transcription factor 16) is a negative regulator that inhibits adipocyte differentiation. AIM:This study was conducted to determine the role of Klf16 in adipocyte differentiation in the context of the SMD pathway.Methods: 3T3-L1 cells were induced and cultured in vitro by cocktail method, Knockdown and Overexpression of STAU1 and KLF16. Then, adipocyte differentiation andexpression of adipogenic-related genes (STAU1, KLF16, PPARγ, and Lipin1) were measured by RT-qPCR and Western blot.RNA immunoprecipitation (RIP) method verified that STAU1 protein can bind to KLF16.Results: The results revealed that STAU1 regulates Klf16 expression at the post-transcriptional level during the adipogenic differentiation of 3T3-L1 cells.STAU1 candirectly bind the 3′UTR of Klf16 mRNA. Klf16 mRNA was found to be degraded through the SMD pathway, thus promoting adipocyte differentiation.Conclusions: In this study, the mechanism of adipocyte differentiation regulation at the post-transcriptional level is demonstrated, and Klf16 is shown as a substrate of the SMD pathway, thus providing new insights into adipogenesis.