<p>SOCS2 is known to regulate myoblast differentiation and skeletal muscle development via the GH-IGF1 axis, while its role in Hu sheep myoblast differentiation and its potential alternative signaling pathways remain largely unexplored. Proteomic profiling of SOCS2-knockout C2C12 monoclonal cell lines identified significant enrichment of differentially expressed proteins in the proteasome pathway, with PSMB9 serving as one of the most prominent candidates, though the underlying mechanism remained unclear. In this present study, the regulatory relationship between SOCS2 and PSMB9 was firstly validated, and the results showed that SOCS2 positively regulated PSMB9 expression in Hu sheep myoblasts. Functional assays further confirmed SOCS2 negatively regulated Hu sheep myoblast differentiation. Mechanistically, JASPAR database predicted potential STAT3 transcription factor binding sites in the PSMB9 promoter region, and dual-luciferase reporter assays verified that STAT3 indeed repressed PSMB9 transcriptional activity. SOCS2 regulated STAT3 protein abundance and phosphorylation by interacting with JAK1. Functionally, STAT3 promoted Hu sheep myoblast differentiation, whereas PSMB9 inhibited Hu sheep myoblast differentiation. Moreover, either STAT3 knockdown or PSMB9 overexpression could rescue the enhanced differentiation phenotype induced by SOCS2 knockdown. Collectively, this study demonstrates a novel mechanism by which SOCS2 regulates Hu sheep myoblast differentiation through STAT3/PSMB9 signaling axis.</p>