Abstract Obesity is strongly associated with metabolic diseases, which have become a global health problem. Exploring the underlying mechanism of adipogenesis is crucial for the treatment of excess white fat. Oncogene YBX1 is a multifunctional DNA- and RNA-binding protein that regulates brown adipogenesis. However, the role of YBX1 in white adipogenesis and adipose tissue expansion remains unknown. Here, we showed that YBX1 deficiency inhibited murine and porcine adipocyte differentiation. YBX1 positively regulated adipogenesis through promoting ULK1- and ULK2-mediated autophagy. Mechanistically, we identified YBX1 serves as a 5-methylcytosine (m5C)-binding protein directly targeting m5C-containing Ulk1 mRNA by using RNA immunoprecipitation. RNA decay assay further proved that YBX1 upregulated ULK1 expression though stabilizing its mRNA. Meanwhile, YBX1 promoted Ulk2 transcription and expression as a transcription factor, thereby enhancing autophagy and adipogenesis. Importantly, YBX1 overexpression in white fat enhanced ULK1/ULK2-mediated autophagy and promoted adipose tissue expansion in mice. Collectively, these findings unveil the post-transcriptional and transcriptional mechanism and functional importance of YBX1 in autophagy and adipogenesis regulation, providing an attractive molecular target for therapies of obesity and metabolic diseases.