Objective To investigate the differentiation of vascular smooth muscle cells (VSMCs) during calcification, and to seek the target for the treatment of cardiovascular calcification. Methods Animal experiment: C57BL/6J mice were randomly assigned to control group (CTL group, fed with normal diet for 4 weeks) or calcification group (CM group, fed with high-fat and high-purine diet for 4 weeks, intraperitoneal injection of 10 mg/kg vitamin D for 2 weeks), with 3 mice in each group. Single-cell transcriptome sequencing was used to evaluate the gene expression of mouse aortic smooth muscle cells. Cell experiment: the primary rat smooth muscle cells were assigned to CTL group (cultured in DMEM containing 10% fetal bovine serum and 1% penicillin) or CM group (cultured in DMEM containing 10 mmol/L sodium glycerophosphate and 3 mmol/L CaCl₂). The gene expression of smooth muscle cells was evaluated by quantitative polymerase chain reaction and Western blotting. Results When smooth muscle cells changed from contractile phenotype to osteoblast phenotype, 13 new cell subtypes were identified by single-cell transcriptome sequencing. Differential gene analysis of smooth muscle cells between the 2 groups showed that the expression of G protein signaling modulator 2 (GPSM2)⁺ smooth muscle cells changed the most. Pseudotime trajectory analysis showed that during the calcification process, GPSM2⁺smooth muscle cell clusters may transform into sphingosine-1-phosphate receptor 3 (S1PR3)⁺ cell clusters, suggesting the change characteristics of smooth muscle cells in calcification environment. Kyoto Encyclopedia of Genes and Genomes analysis of S1PR3⁺ smooth muscle cell clusters showed that mitogen-activated protein kinase (MAPK) signaling pathway was activated during calcification. Western blotting analysis further demonstrated that calcification environment stimulated extracellular signal-regulated kinase (ERK)1/2 phosphorylation. Conclusion Under calcification condition, vascular smooth muscle cells transform from the GPSM2⁺ smooth muscle cell cluster to the S1PR3⁺ smooth muscle cell cluster, suggesting that the S1PR3⁺ smooth muscle cell cluster may serve as a target for the treatment of cardiovascular calcification.