Previous studies have reported that theN6-methyladenosine demethylase ALKBH5 can regulate adipogenesis in humans. However, its function in birds remains unclear. In this study we aimed to explore the expression and function of theALKBH5gene in chicken adipose tissue. The results showed thatALKBH5is widely expressed in various chicken tissues, and the expression ofALKBH5is relatively higher in abdominal adipose tissue. In addition, the expression ofALKBH5in abdominal adipose tissue of lean broilers is higher than that in fat broilers at 2 and 3 weeks of age. Moreover, the proliferation and differentiation of preadipocytes are associated with reduced and increased expression ofALKBH5, respectively. We also found thatALKBH5knockout promotes preadipocyte proliferation, as evidenced by an increase in cell viability, DNA replication activity, G1-S cell cycle progression, and the expressions ofKi67andPCNA. Furthermore, after knockout ofALKBH5, the lipid droplet accumulation and the expression ofPPARγ,A-FABP, andFASare reduced significantly. Thus, our results indicated that ALKBH5 is a novel regulator of proliferation and differentiation of chicken preadipocytes.