Clinically, chronic pain and hyperalgesia induced by muscle injury are disabling and difficult to treat. Cellular and molecular mechanisms underlying chronic muscle-induced hyperalgesia are not well understood. For this reason, we developed an animal model where repeated injections of acidic saline into one gastrocnemius muscle produce bilateral, long-lasting mechanical hypersensitivity of the paw (i.e. hyperalgesia) without associated tissue damage. Since acid sensing ion channels (ASICs) are found on primary afferent fibers and respond to decreases in pH, we tested the hypothesis that ASICs on primary afferent fibers innervating muscle are critical to development of hyperalgesia and central sensitization in response to repeated intramuscular acid. Dorsal root ganglion neurons innervating muscle express ASIC3 and respond to acidic pH with fast, transient inward and sustained currents that resemble those of ASICs. Mechanical hyperalgesia produced by repeated intramuscular acid injections is prevented by prior treatment of the muscle with the non-selective ASIC antagonist, amiloride, suggesting ASICs might be involved. ASIC3 knockouts do not develop mechanical hyperalgesia to repeated intramuscular acid injection when compared to wildtype littermates. In contrast, ASIC1 knockouts develop hyperalgesia similar to their wildtype littermates. Extracellular recordings of spinal wide dynamic range (WDR) neurons from wildtype mice show an expansion of the receptive field to include the contralateral paw, an increased response to von Frey filaments applied to the paw both ipsilaterally and contralaterally, and increased response to noxious pinch contralaterally after the second intramuscular acid injection. These changes in WDR neurons do not occur in ASIC3 knockouts. Thus, activation of ASIC3s on muscle afferents is required for development of mechanical hyperalgesia and central sensitization that normally occurs in response to repeated intramuscular acid. Therefore, interfering with ASIC3 might be of benefit in treatment or prevention of chronic hyperalgesia.