Endothelial thrombomodulin (TM) is capable of causing thrombin-dependent degradation of high mobility group box 1 (HMGB1), a damage-associated molecular pattern protein. We have shown that HMGB1 is involved in chemotherapy-induced peripheral neuropathy (CIPN) and visceral pain, which are prevented by recombinant soluble TM (TMα). Most interestingly, anticoagulants abolish the anti-CIPN effect of TMα, and enhance the severity of CIPN through increased blood HMGB1 levels. To clarify the role of endogenous TM/thrombin system in visceral pain modulation, in the present study, we tested the effects of argatroban, a thrombin inhibitor, on HMGB1-dependent colonic and bladder pain in mice. TMα reversed the butyrate (Bu)-induced colonic hypersensivity, an effect abolished by argatroban, and repeated treatment with argatroban promoted the colonic hypersensitivity in mice treated with a subeffective dose of Bu. Similarly, TMα prevented the cyclophosphamide (CPA)-induced bladder pain, an effect abolished by argatroban, and argatroban promoted the bladder pain in mice treated with a subeffective dose of CPA. Our data suggest that anticoagulants aggravate HMGB1-dependent visceral pain most probably by inhibiting the endothelial TM/thrombin system capable of degrading HMGB1.