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AIM This study aimed to investigate the effect of 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) on primary chondrocytes cultured from patients with osteoarthritis (OA). METHOD Primary chondrocytes isolated from the tibial plateau of female OA patients were characterized by immunocytochemistry analysis. Using Cell Counting Kit-8 (CCK-8), cell viability was measured to select suitable 1,25(OH)2D3 concentrations for treating chondrocytes. RNA-sequencing was performed on primary chondrocytes treated with or without 1,25(OH)2D3. Differentially expressed genes (DEGs) as well as gene ontology (GO)-biological process (BP) and pathways affected by 1,25(OH)2D3 were identified. Protein-protein interaction (PPI) network was constructed, and the hub nodes in the PPI network were identified. qRT-PCR was conducted to confirm the expression levels of six DEGs. RESULTS Positive collagen II staining confirmed the successful isolation of primary chondrocytes. CCK-8 assay showed maximal primary chondrocyte survival rate when treated with 10-5 μmol/L of 1,25(OH)2D3 for 72 h. RNA-sequencing results identified a total of 1036 DEGs, including 593 upregulated and 443 downregulated genes from 1,25(OH)2D3 treated and untreated cells. Further functional enrichment analyses showed the association of these DEGs with GO-BP terms such as response to the stimulus, cell proliferation, angiogenesis, and regulation of cell motility, and KEGG pathways, including TNF signaling pathway, IL-17 signaling pathway, cytokine-cytokine receptor interaction, and NF-kappa B signaling pathway. PPI network identified UBC, FOS, IFIT1, CDK1, and ISG15 as the hub nodes in the network. qRT-PCR results were in alignment with the results of RNA-sequencing. CONCLUSION Our study might provide a theoretical basis for the use of vitamin D in treating OA.