Aerobic glycolysis, also known as Warburg effect, is a key hallmark of cancers. The Y-box-binding protein 1 (YBX1) is a well-known oncoprotein implicated in multiple malignant phenotypes of cancers. Meanwhile, little is known about the oncogenic functions and mechanisms of YBX1 in bladder cancer. Based on gene set enrichment analysis (GSEA) of TCGA RNAseq data, we find that YBX1 was profoundly involved in the glycolysis part of glucose metabolism. Loss- and gain-of-function studies show that YBX1 can enhance glycolysis as revealed by expression of glycolytic enzymes, glucose uptake, lactate secretion and extracellular acidification rate (ECAR). Inhibition of glycolysis completely compromises the tumor-promoting effect of YBX1 on tumor growth. Mechanistically, YBX1 regulates the expression of c-Myc and HIF1α, which further upregulate glycolytic enzymes to facilitate glycolysis. Moreover, in vivo study further confirms that genetic silencing of YBX1 markedly attenuates tumor growth and this tumor-suppressive effect is largely dependent on reduced glycolysis. Taken together, these results, as a proof of principle, provide a novel insight into the oncogenic role of YBX1 in glycolysis and suggest the potential therapeutic strategy by targeting YBX1 in bladder cancer.