Bladder pain accompanying cyclophosphamide (CPA)-induced cystitis involves two pathways: 1) upregulation of cystathionine-γ-lyase (CSE) that generates H₂S, which enhances Ca_v3.2 T-type Ca²⁺ channel activity (Br J Pharmacol 2012;167:917), and 2) RAGE activation by HMGB1, a DAMP protein (Neuropharmacology 2014;79:112). We thus analyzed molecular mechanisms underlying a possible crosstalk of those signaling cascades. The CPA-induced bladder pain and CSE upregulation in mice were suppressed by an anti-HMGB1-neutralizing antibody, RAGE antagonist or macrophage (Mφ) depletor. Mφ accumulation was detected in the bladder following CPA treatment. Acrolein, a hepatic metabolite of CPA, evoked prompt ATP release from cultured human urothelial T24 cells. ATP induced HMGB1 release from mouse Mφ-like RAW264.7 cells, an effect blocked by A438079, a P2X₇ antagonist, and by inhibitors of NF-κB or p38MAPK and an antioxidant. A438079 also attenuated CPA-induced bladder pain in mice. These data suggest that urothelium-derived ATP evokes ROS-dependent HMGB1 release from Mφ via P2X₇, which in turn causes RAGE-dependent CSE upregulation and then H₂S/Ca_v3.2 signaling essential for bladder pain.