Radiofrequency (RF) fields have been classified as probable human carcinogens and non-thermal RF-radiation (RFR) can cause an increase in oxidative stress and inflammation in tissues. Vitamin D has been researched for many years for its anti-cancer and suppressive effects on cancer cell growth. In this study, we aimed to examine the effects of exposure to RF signals on cell viability, free radical formation, and related inflammation, and the restorative effect of vitamin D against this. For this purpose, MCF-7 and MDA-MB-231 cells were exposed to 2.1GHz W-CDMA-modulated RFR. The viability of cells was determined by MTT assay, and the percentage of apoptotic cells was determined by FITC-conjugated Annexin-V/PI assay. The expression levels of VDR, NF- κB, COX-2, and p53 genes were studied using Real-Time-PCR. ROS analysis was performed by flow cytometry. A statistically significant decrease in MCF-7 cell viability (p < 0.001) was observed with increasing doses of calcitriol compared to the control group, while no significant difference was observed in MDA-MB-231 cells (p > 0.05). As a result of ROS analysis, significant decreases in life values were observed for both cells due to calcitriol. A statistically significant increase in p53 expression was observed with increasing calcitriol dose in both cell lines. An increase in VDR, NF-kappa B, and COX2 expressions was also observed in MCF-7 cells. We observed that calcitriol administered after RF exposure caused an increase in apoptosis and mRNA expression of inflammation markers, a decrease in ROS levels, and an increase, and this can be evaluated in cancer treatment or prevention.