The exact mechanism of the masseter muscle pain recognized as a prominent symptom in temporomandibular disorders remains unclear, although it is clinically known that excessive muscular contraction causes tenderness in masseter muscles. It has been demonstrated that P2X3 receptors (P2X3Rs) in sensory neurons play a role in pain signaling from the periphery. We determined the role of P2X(3)R on pressure pain and mechanical hyperalgesia in a newly developed rat model of masseter muscle pain. The pain in the masseter muscle was assessed by the pressure pain threshold (PPT), which was defined as the amount of pressure required to induce head flinching. In naive animals, systemic treatment with morphine was associated with increase of PPTs. Changes in PPTs were examined after administration of P2XR agonists or antagonists into the masseter muscle. The masseter muscle injection of alpha,beta-meATP (P2X(1,3,2/3)R-specific agonist) induced a significantly greater behavioral response than its vehicle. This enhanced response was completely blocked by the co-application of alpha,beta-meATP with PPADS (P2X(1,2,3,5,1/5,2/3)R-specific antagonist). Excessive contraction in masseter muscle was produced by electrical stimulation. The exerted masseter muscles showed a significant reduction in PPTs indicating the induction of mechanical hyperalgesia of the muscle. Moreover, administration of PPADS to the exerted masseter muscles produced a complete recovery of reducing PPT. Immunohistochemically, the number of P2X3R-positive neurons innervating the masseter muscles increased in the excessively contracted condition in trigeminal ganglia. Our results suggested that P2X3R plays an important role in pressure pain and mechanical hyperalgesia in masseter muscle caused by excessive muscular contraction.