Bladder pain accompanying cyclophosphamide (CPA)-induced cystitis in mice involves the upregulation of cystathionine-γ-lyase (CSE), an H2S-forming enzyme, followed by increased activity of Cav3.2 T-type Ca2+ channels, known as targets for H2S [1], and also extracellular high mobility group box 1 (HMGB1) targeting the receptor for advanced glycation end products (RAGE) [2]. We thus analyzed the relationship between the HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in bladder pain signaling. Female ddY mice (16-24 g) received i.p. CPA at 400 mg kg-1. As reported previously [1], bladder pain-like behavior (BP) was counted from 3.5 to 4 h after CPA treatment, and thereafter, referred hyperalgesia (RH) in the lower abdomen was evaluated by determining nociceptive scores following stimulation with von Frey hairs. After cervical dislocation, the bladder was isolated for measurement of bladder weight (BW) and Western blot analysis of CSE. Data show the mean ± SEM. Statistical significance was analyzed by Kruskal-Wallis H-test followed by a least significant difference-type test for nociceptive score and by ANOVA followed by Tukey’s test for all other data. The anti-HMGB1 antibody (Ab) at 1 mg kg-1, administered i.p., prevented CPA-induced BP [vehicle (V)+V 2.4±1.2, control IgG (IgG)+CPA 35.4±4.8 (p<0.01 vs. V+V), Ab+CPA 13.3±1.8 (p<0.01 vs. IgG+CPA), n=5-6] and RH [total nociceptive score (TNS) from 10 challenges with 0.07 g hair: V+V 6.2±0.58, IgG+CPA 11.4±0.4 (p<0.001 vs. V+V), Ab+CPA 8.1±0.6 (p<0.05 vs. IgG+CPA), n=5-6], but not BW increase, an indicator of swelling [BW (mg g-1 body weight): V+V 0.8±0.04, IgG+CPA 1.77±0.19 (p<0.01 vs. V+V), Ab+CPA 1.89±0.07 (n.s. vs. IgG+CPA), n=4-6]. The CPA-induced upregulation of bladder CSE was also significantly reduced by Ab [V+V 0.057±0.016, IgG+CPA 0.98±0.033 (p<0.01 vs. V+V), Ab+CPA 0.70±0.12 (p<0.05 vs. IgG+CPA), n=5]. Liposomal clodronate (LClo) (1.05 mg per mouse) that depletes macrophages (Mφ) prevented the CPA-induced BP [control liposome (cL)+V 0.83±0.48, cL+CPA 39.33±5.06 (p<0.01 vs. cL+V), LClo+CPA 12.5±3.12 (p<0.01 vs. cL+CPA), n=6], RH [TNS: cL+V 5.3±0.61, cL+CPA 13.5±0.76 (p<0.001 vs. cL+V), LClo+CPA 8.6±0.95 (p<0.05 vs. cL+CPA), n=6] and CSE upregulation [cL+V 0.095±0.045, cL+CPA 0.62±0.036 (p<0.01 vs. cL+V), LClo+CPA 0.36±0.095 (p<0.05 vs. cL+CPA), n=5]. FPS-ZM1 (FPS), a RAGE antagonist, preadministered at 1 mg kg-1, suppressed the CPA-induced BP [V+V 1.0±0.63, V+CPA 37.6±2.9 (p<0.01 vs. V+V), FPS+CPA 12.1±1.3 (p<0.01 vs. V+CPA), n=6], RH [TNS: V+V 3.8±0.75, V+CPA 12.3±0.42 (p<0.001 vs. V+V), FPS+CPA 8.5±0.62 (p<0.05 vs. V+CPA), n=6] and CSE upregulation [V+V 0.11±0.047, V+CPA 1.1±0.10 (p<0.01 vs. V+V), FPS+CPA 0.74±0.14 (p<0.05 vs. V+CPA), n=6]. Thus, Mφ-derived HMGB1 is considered to mediate CSE upregulation via RAGE during cystitis, leading to H2S-dependent bladder pain.