The present study was undertaken to evaluate the synergistic effect of combining hyperthermia with irradiation and calcium carbonate nanoparticles (CC NPs) on proliferation of MCF-7 cells. The cells were randomly allocated to 19 groups: one negative control, three positive controls and 15 treatment groups. MCF-7 cells were treated with three concentrations of CC NPs (50, 100 and 150 μg/mL), gamma radiation (200 cGy), hyperthermia (41 °C for 1 h) and three concentrations of doxorubicin (200, 400 and 800 nm) and incubated at 37 °C for 24 h. Then the cell viability, the percentage of apoptosis and the levels of caspase-3, -8 and -9 proteins were measured. The results indicated that the combination group (150 µg/mL CC NPs + thermoradiotherapy) had a significant (p < .001) decrease in cell viability (48.65 ± 4.8%) and a significant (p < .001) increase in apoptosis percentage (45 ± 1.63%) of MCF-7 cells, as compared with the negative control and most of the other treatment groups. Moreover, a significant (p < .05) increase was observed in the activity of caspase-3 and caspase-9. Our findings revealed that CC NPs in combination with irradiation and hyperthermia could significantly reduce the cell viability and enhance the apoptosis of the MCF-7 breast cancer cells, the same as doxorubicin anti-cancer drug.