INTRODUCTION: Vascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways. MATERIALS AND METHODS AND RESULTS: Human coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium phosphate medium (CaP) to induce a mineralized extracellular matrix. OM induces osteoblast-like differentiation of SMCs-a key process in intimal calcification during atherosclerotic plaque remodeling. CaP mimics hyperphosphatemia, associated with chronic kidney disease-a risk factor for medial calcification. Transcriptomic analysis revealed distinct gene expression profiles of OM and CaP-calcifying SMCs. OM and CaP-treated SMCs shared 107 differentially regulated genes related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated decreased mitochondrial respiration and glycolysis in CaP-treated SMCs compared to increased mitochondrial respiration without altered glycolysis in OM-treated SMCs. Subsequent kinome and drug repurposing analysis (Connectivity Map) suggested a distinct role of protein kinase C (PKC). validation experiments demonstrated that the PKC activators prostratin and ingenol reduced calcification triggered by OM and promoted calcification triggered by CaP. CONCLUSION: Our direct comparison results of two calcification models strengthen previous observations of distinct intracellular mechanisms that trigger OM and CaP-induced SMC calcification . We found a differential role of PKC in OM and CaP-calcified SMCs providing new potential cellular and molecular targets for pharmacological intervention in VC. Our data suggest that the field should limit the generalization of results found in studies using different calcification protocols.