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Objectives: RNF20 is recognized as a main E3 ligase for monoubiquitination of histone H2B at lysine 120 (H2Bub). The critical role of RNF20 and H2Bub in various molecular events, such as DNA replication, RNA transcription, and DNA damage response, has been widely investigated and documented. However, its role in porcine adipogenesis remains unknown. In this study, we aimed to clarify the effect of RNF20 on porcine preadipocyte differentiation. Materials and Methods: Backfat tissues from fat‐type pigs (Bama and Meishan) and lean‐type pigs (Yorkshire and Landrace) were collected to detect the expression level of RNF20. Preadipocytes were isolated from Bama piglets and induced to differentiation. Small interfering RNAs were applied to deplete RNF20. Oil Red O staining, quantitative real‐time PCR, RNA‐seq, Western blot analysis, and EdU assays were performed to study the regulatory mechanism of RNF20 during adipogenesis. Results: We found that the expression levels of RNF20 and H2Bub were significantly higher in backfat tissues from fat‐type pigs than in those from lean‐type pigs. Consistently, the significantly induced expression of RNF20 and H2Bub was also observed in porcine differentiated adipocytes. In addition, knockdown of RNF20 greatly inhibited porcine adipogenesis, as evidenced by dramatically decreased lipid droplet formation and lower expression levels of adipogenic transcription masters in RNF20 knockdown cells. Mechanistically, the depletion of RNF20 decreases the cell proliferation and the level of p‐C/EBPβ via the Ras‐Raf‐MEK1/2‐ERK1/2 cascade pathway at the mitotic clonal expansion phase and therefore suppresses cell differentiation. Conclusions: Our results demonstrate that RNF20 is required for porcine preadipocyte differentiation. [ABSTRACT FROM AUTHOR]