Disruption of the fatty acid oxidation process (FAO) significantly affects the tumorigenesis of bladder cancer (BC). Apart from energy regulation, acetyl-CoA from FAO, as an important substrate for protein acetylation, plays an important role in BC. Here, we found that long-chain fatty acid synthase 5 (ACSL5) acting as a key enzyme in the initial stage of FAO, was downregulated in BC, and the decreased level of ACSL5 was strongly associated with a poor prognosis for BC patients. We found that the main reason for the low expression of ACSL5 in BC is the highly methylated CpG islands in its DNA, which is closely regulated by DNA methyltransferase 1 (DNMT1). Upregulating ACSL5 expression in BC cells significantly promoted FAO, and reduces the intracellular lipid content while increasing the level of acetyl-CoA. We demonstrated that restoration of ACSL5 expression promoted K1360 acetylation of TP53-binding protein 1 (53BP1), through posttranslational modification (PTM), subsequently enhancing the recruitment of the P53-P21 senescent signaling axis in the nucleus and promoting cellular senescence. In vivo and in vitro, ACSL5 overexpression promoted BC senescence and inhibited BC cell proliferation, and elaidic acid (EA) feeding further enhanced these effects. In summary, our study revealed that ACSL5-mediated lipid oxidation reduces the intracellular lipid content, promotes cellular senescence, and inhibits the proliferation of BC. The activation of ACSL5-mediated lipid oxidation to regulate cellular senescence may provide an innovative direction for BC therapy.