Neuronal proton-gated acid-sensing ion channels (ASICs) participate in the detection of tissue acidosis, a phenomenon often encountered in painful pathologic diseases. Such conditions often involve in parallel the activation of various signaling pathways such as mitogen activated protein kinases (MAPKs) that ultimately leads to phenotype modifications of sensory neurons. Here, we identify one member of the MAPKs, c-Jun N-terminal kinase (JNK), as a new post-translational positive regulator of ASICs in rodent sensory neurons. Recombinant H+-induced ASIC currents in HEK293 cells are potently inhibited within minutes by the JNK inhibitor SP600125 in a subunit-dependent manner, targeting both rodent and human ASIC1b and ASIC3 subunits (except mouse ASIC3). The regulation by JNK of recombinant ASIC1b- and ASIC3-containing channels (homomers and heteromers) is lost on mutation of a putative phosphorylation site within the intracellular N- and the C-terminal domain of the ASIC1b and ASIC3 subunit, respectively. Moreover, short-term JNK activation regulates the activity of native ASIC1b- and ASIC3-containing channels in rodent sensory neurons and is involved in the rapid potentiation of ASIC activity by the proinflammatory cytokine TNFα. Local JNK activation in vivo in mice induces a short-term potentiation of the acid-induced cutaneous pain in inflammatory conditions that is partially blocked by the ASIC1-specific inhibitor mambalgin-1. Collectively, our data identify pain-related channels as novel physiological JNK substrates in nociceptive neurons and propose JNK-dependent phosphorylation as a fast post-translational mechanism of regulation of sensory-neuron-expressed ASIC1b- and ASIC3-containing channels that may contribute to peripheral sensitization and pain hypersensitivity.