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) mu mol/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.