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Obesity presents a serious threat to human health and broiler performance. The expansion of adipose tissue is mainly regulated by the differentiation of preadipocytes. The differentiation of preadipocytes is a complex biological process regulated by a variety of transcription factors and signaling pathways. Previous studies have shown that the transcription factor HMG-box protein 1 (HBP1) can regulate the differentiation of mouse 3T3-L1 preadipocytes by activating the Wnt/β-catenin signaling pathway. However, it is unclear whether HBP1 involved in chicken preadipocyte differentiation and which signaling pathways it regulates. The aim of the current study was to explore the biological function and molecular regulatory mechanism of HBP1 in the differentiation of chicken preadipocytes. The expression patterns of chicken HBP1 in abdominal adipose tissue and during preadipocyte differentiation were analyzed by RT-qPCR and Western blot. The preadipocyte stably overexpressing HBP1 or knockout HBP1 and their control cell line were used to analyze the effect of HBP1 on preadipocyte differentiation by oil red O staining, RT-qPCR and Western blot. Cignal 45-Pathway Reporter Array was used to screen the signal pathways that HBP1 regulates in the differentiation of chicken preadipocytes. Chemical inhibitor and siRNA for signal transducer and activator of transcription 3 (STAT3) were used to analyze the effect of STAT3 on preadipocyte differentiation. The preadipocyte stably overexpressing HBP1 was transfected by the siRNA of STAT3 or treated with a chemical inhibitor of STAT3 for the rescue experiment. The results of gene expression analysis showed that the expression of HBP1 was related to abdominal fat deposition and preadipocyte differentiation in chickens. The results of function gain and loss experiments indicated that overexpression/knockout of HBP1 in chicken preadipocytes could inhibit/promote (P<0.05) lipid droplet deposition and the expression of adipogenesis-related genes. Mechanismlly, HBP1 activates (P<0.05) the signal transducer and activator of transcription 3 (STAT3) signaling pathway by targeting janus kinase 2 (JAK2) transcription. The results of functional rescue experiments indicated that STAT3 signaling mediated the regulation of HBP1 on chicken preadipocyte differentiation. In conclusion, HBP1 inhibits chicken preadipocyte differentiation by activating the STAT3 signaling pathway viadirectly enhancing JAK2expression. Our findings provided new insights for further analysis of the molecular genetic basis of chicken adipose tissue growth and development.