Piezo1, a mechanosensitive protein, has been implicated in the regulation of osteogenesis. Here we aimed to elucidate the role of Piezo1 in mechanical-induced endochondral osteogenesis at temporomandibular joint (TMJ) by investigating Piezo1-specific inactivation in Col2α1-expressing cell lineages. Piezo1flox/flox; Col2α1-Cre+(Piezo1Col2α1KO) mice were generated for in vivo study. A mandibular advancement (MA) model was established to induce endochondral osteogenesis in the TMJ condyle. Subchondral bone quality was assessed using micro-CT, while changes in the distribution and expression levels of Piezo1, Ocn, and Osx were evaluated via immunofluorescence staining. In vitro, ATDC5 cells were subjected to 6% cyclic tensile strain (CTS) using a Flexcell device. The effects of Piezo1 inactivation under CTS were analyzed using real-time PCR and western blotting. Piezo1, Ocn, and Osx were overexpressed in the MA-induced condylar cartilage and CTS-treated ATDC5. Piezo1 inactivation inhibited the mechanical-induced endochondral osteogenesis of TMJ condylar in mice. Piezo1 silencing inhibited the osteogenic expression as well as the PI3K/Akt pathway under CTS, which could be rescued by the application of Akt activator, SC79. Piezo1 is a critical regulator of mechanical-induced endochondral osteogenesis in TMJ condylar. Chondrocyte-specific Piezo1 inactivation under tensile loading impairs the quality of newly formed bone in vivo, while increase of osteogenic marker was downregulated through PI3K/Akt signaling in vitro.