The members of the degenerin/epithelial sodium channels (DEG/ENaCs) family have been demonstrated as essential mechanotransducers in the nematode Caenorhabditis elegan and fly Drosophila malanogaster. As a mammalian homologue, the proton-gated, amiloride-sensitive acid-sensing ion channel 3 (ASIC3) may thus play a role in mechanosensation. To map the peripheral innervations of ASIC3-expressing sensory neurons, we first knock-in a membrane-tagged farnesylated enhanced green fluorescent protein (eGFP-f) into the mouse Accn3 allele. In the eGFP-f knock-in mice, immunostaining of GFP indicated that ASIC3-containig nerves are projected to the gastrocnemius muscle, whisker hair root, and colon wall, all tissues with strong indication of mechanosensory requirement. To achieve DRG cell type-specific elimination of ASIC3, we generated another floxed allele of mouse Accn3 for Cre-mediated conditional knockout. We used Nav-1.8-Cre and Parvalbumin-Cre mice to selectively target ASIC3 in nociceptors and proprioceptors, respectively. PCR with genomic DNA and Q-PCR suggested that the floxed Accn3 exon1 is faithfully excised in Cre-expression tissues. Seeding neurons on an elastic substrate, polydimethylsiloxane (PDMS), coated with fibronectin, we then performed whole-cell patch clamping to record the stretch-activated action potentials to probe the mechano-sensing property. In the wildtype DRG proprioceptors, mechanical stretch on the neurite induced action potentials, and this phenomenon was disappeared in a large population of ASIC3-null proprioceptors. Finally, we report behavioral phenotype between these mice in the beam-walking task, a well-characterized proprioception test for muscle spindle Ia afferent defect. Combined these data, we provided strong evidence that ASIC3 is essential for mechanosensation of muscle stretch, the sense of proprioception.