To develop a rodent model of persistent non-inflammatory bladder pain and to test macrophage migration inhibitory factor and high mobility box group 1 as mediators of bladder pain. Female C57BL/6 mice received intravesical instillations of protease activated receptor 4 (100 μmol/L, for 1 h) three times every other day and abdominal mechanical hypersensitivity (50% mechanical threshold) was tested on day 0 (baseline), and at days 1, 2, 3, 4, 7 and 9 after the first protease-activated receptor 4 injection. At the end of the experiment, micturition changes were measured and bladders were examined for histological changes. Macrophage migration inhibitory factor antagonist (MIF098; 40 mg/kg i.p. b.i.d.) or high mobility group box 1 inhibitor (glycyrrhizin; 50 mg/kg i.p. daily) was administered from day 2 until day 8. There was a significant and persistent decrease in abdominal mechanical threshold starting from day 3 in the protease-activated receptor 4-treated group that persisted until day 9 (5 days post-last instillation), but not in the control group. Glycyrrhizin fully reversed while MIF098 partially reversed abdominal mechanical hypersensitivity in protease-activated receptor 4-treated mice. The changes started on day 3 after the first protease-activated receptor 4 instillation, and analgesic effects lasted throughout the rest of the testing period. None of the groups had significant micturition changes or overt bladder histological changes. Repeated intravesical protease activated receptor 4 instillations produce persistent bladder pain without inflammation. Macrophage migration inhibitory factor and high mobility group box 1 are possible effective target molecules for bladder pain alleviation.