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The highly regulated proliferation of adipocytes plays a momentous role in fat development and obesity. Hoxa5 is an important member of the Hox family, its encoded protein is an important transcription factor related to development, and its differential expression in different adipose tissues seems to indicate that Hoxa5 may be involved in the regulation of adipocyte proliferation. To evaluate the regulatory mechanism of Hoxa5 on adipocyte proliferation, we constructed a variety of Hoxa5 expression vectors in vivo and in vitro to explore its mechanism on adipocyte proliferation and its potential impact on obesity. We observed that the overexpression of Hoxa5 strongly reduces cell counts and Hoxa5 can inhibit cell proliferation and block cell cycle progression by regulating the expression of genes such as Cyclin E, Cyclin D1, and p53. Most importantly, we demonstrated that Hoxa5 exerts its effect by regulating the signaling pathway of Janus kinase 2 (JAK2) signal transduction and transcription 3 (STAT3) activator, as well as binding to the promoter region of Ccne1 and inhibiting the transcription of Ccne1. This study provides an in-depth understanding of the potential molecular mechanism of Hoxa5 inhibiting adipocyte proliferation. Our results suggest the importance of Hoxa5 in the treatment of obesity. [ABSTRACT FROM AUTHOR]