BACKGROUND: Switching from a contractile to a synthetic phenotype of pulmonary arterial smooth muscle cells (PASMCs) is known to play a crucial role in pulmonary arterial hypertension (PAH). We investigated how hypoxia and mechanical stress mediate the phenotypic switching of PASMCs. METHODS: Human PASMCs were used for experiments. Hypoxia treatment was done by culturing cells under 1% O₂. Mechanical stress was induced by loading cells to 50 mmHg hydrostatic pressure. We analyzed cell morphology, cell proliferation, phenotypic marker protein expression, cytokine release, and the activation of stress-related pathways at 24 h after treatment. Bulk and single-cell RNA-sequencing datasets were used to analyze heat shock protein family D member 1 (HSPD1) expression in PAH lungs and PASMCs. Heat shock protein 60 (HSP60) was knocked down in PASMCs by transfection of HSPD1-siRNA. RESULTS: Either hypoxia or mechanical stress alone induced the morphology change, increased cell proliferation, and promoted the phenotypic switching and inflammatory cytokines release of PASMCs. Interestingly, all those were dramatically enhanced under the combination of hypoxia and mechanical stress. Mechanistically, we found that the combination of hypoxia and mechanical stress not only significantly enhanced the mitochondrial HSP60 expression but also induced its partial redistribution to the cytosol. Bioinformatic analyses also confirmed the elevated HSPD1 expression in PAH lungs and PASMCs. HSP60 knockdown effectively attenuated the phenotypic switching of PASMCs induced by hypoxia and mechanical stress. CONCLUSION: Hypoxia- and mechanical stress-induced upregulation of mitochondrial HSP60 is associated with phenotypic switching of PASMCs.