The uremic toxin indoxyl sulfate (IS) is known to accumulate in chronic kidney disease (CKD) patients and harm the renal tubular cells. Transient receptor potential vanilloid 1 (TRPV1) is present in the kidney and acts as a renal sensor. However, the mechanism underlying IS-mediated renal tubular damage in view of TRPV1 is lacking. We showed that hyperfunction of calcium-permeable TRPV1 contributes to the IS-mediated tubulotoxicity. IS activated aryl hydrocarbon receptor (AhR) and then upregulated 12-lipoxygenase (ALOX12), and this induced endovanilloid 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) synthesis and contributed to TRPV1 hyperfunction. These indicate that calcium overload participates in the IS-induced cell damage.