Titanium dioxide nanoparticles (TiO2 NPs) can cause apoptosis in TM4 cells; however, the underlying mechanism has not been entirely elucidated. The purpose of this study was to investigate the effects of TiO2 NPs on ROS, Ca2+ level, p38/AKT/mTOR pathway, and apoptosis in TM4 cells and to evaluate the role of Ca2+ in p38/AKT/mTOR pathway and apoptosis. After exposure to different concentrations (0, 50, 100, 150, and 200 μg/mL) of TiO2 NPs for 24 h, cell viability, ROS, Ca2+ level, Ca2+‐ATPase activity, p38/AKT/mTOR pathway‐related proteins, apoptosis rate, and apoptosis‐related proteins (Bax, Bcl‐2, Caspase 3, Caspase 9, and p53) were detected. The ROS scavenger NAC was used to determine the effect of ROS on Ca2+ level. The Ca2+ chelator BAPTA‐AM was used to evaluate the role of Ca2+ in p38/AKT/mTOR pathway and apoptosis. TiO2 NPs significantly inhibited cell viability, increased ROS level, and elevated Ca2+ level while suppressing Ca2+‐ATPase activity. TiO2 NPs regulated the p38/AKT/mTOR pathway via increasing p‐p38 level and decreasing p‐AKT and p‐mTOR levels. TiO2 NPs significantly enhanced the apoptosis. NAC attenuated Ca2+ overload and reduction in Ca2+‐ATPase activity caused by TiO2 NPs. BAPTA‐AM alleviated TiO2 NPs‐induced abnormal expression of p38/AKT/mTOR pathway‐related proteins. BAPTA‐AM assuaged the apoptosis caused by TiO2 NPs. Altogether, this study revealed that TiO2 NPs elevated intracellular Ca2+ level through ROS accumulation. Subsequently, the heightened intracellular Ca2+ level was observed to exert regulation over the p38/AKT/mTOR pathway, ultimately culminating in apoptosis. These results provides a complementary understanding to the mechanism of TiO2 NPs‐induced apoptosis in TM4 cells.