1,25(OH)2D3 has anti‐inflammatory and growth inhibitory effects. Our study explored the effect of 1,25(OH)2D3 treatment on the expression of monocyte chemotactic protein‐1 (MCP‐1), hepatocyte growth factor (HGF), and insulin‐like growth factor‐1 (IGF‐1) by peripheral blood mononuclear cells (PBMCs), peritoneal fluid mononuclear cells (PFMCs), endometrial stromal cells (ESCs), and its effect on the proliferation of PBMCs and PFMCs of patients with endometriosis compared with controls. PBMCs, PFMCs, and ESCs were obtained from 10 endometriosis patients and 10 non‐endometriotic individuals. After treating cells with 0.1 μM of 1,25(OH)2D3 for 6, 24, and 48 h, the gene and protein expression of mentioned factors were evaluated by real‐time PCR and ELISA methods, respectively. 1,25(OH)2D3 treatment significantly reduced the protein expression of MCP‐1, HGF, and IGF‐1 in PBMCs and PFMCs of endometriotic patients at 48 h (p < 0.05–<0.01). Also, this treatment significantly reduced MCP‐1, HGF, and IGF‐1 gene and/or protein expression in EESCs and EuESCs at 24 and 48 h (p < 0.05–<0.01). 1,25(OH)2D3 treatment also reduced the proliferation of PBMCs and PFMCs of endometriotic patients compared with controls (p < 0.01). 1,25(OH)2D3 can be considered as a potentially effective agent in the prevention and treatment of endometriosis along with other therapies.