Derived from mesenchymal stem cells (MSCs) under the influence of various local factors and growth hormones, osteoblasts are one of the leading components of the bone tissue. 1,25-dihydroxyvitamin D3 (calcitriol), an active form of vitamin D, plays an important role in retaining calcium and phosphate homeostasis within the body. Since vitamin D is also known to have effects on cell proliferation, our study aimed to investigate the effects of different concentrations of calcitriol on the proliferation of in vitro cultured osteoblast-like cells from the bone marrow of 90-95 days old young adult rats of both sexes. Calcitriol was applied at six (6) different concentrations (nM): 0.1, 1, 10, 20, 40 i 60. Each concentration was tested four times, counting the cells after 24 and 48 hours. After 24 and 48 hours treatment, the most effective dose of calcitriol for cell proliferation was 0.1 nM for both males and females. The greatest reduction in the frequency of OB-like cells from both sexes after treatment of 24 and 48 hours, was a 60 nM calcitriol concentration. Higher proliferative values of osteoblast-like cells were obtained in males compared to those obtained in females. Thus, the results of the current study reveal that calcitriol treatment induces the proliferation of osteoblast-like cells in a dose-dependent manner and this effect shows difference in cells from different genders.