Serum is a common biomaterial in cell culture that provides nutrients and essential growth factors for cell growth. Serum heat inactivation is a common treatment method whose main purpose is to remove complement factors and viruses. As serum contains many heat-labile factors, heat inactivation may affect cell proliferation, migration, differentiation, and other functions. However, the specific mechanism of its effect on cell function has not been studied. Thus, we investigate the exact effects of heat-inactivated FBS on the viability of various cells and explore the possible molecular mechanisms. We treated HCT116, HT-29, and HepG2 cell lines with heat-inactivated (56 °C for 30 min) medium, DMEM, or fetal bovine serum (FBS) for different times (0, 10, 15, 30, 60, or 90 min); we found that heat-inactivated FBS significantly promoted the viability of these cells, whereas DMEM did not have this effect. Moreover, heat-inactivated FBS stimulated cells to produce a small amount of ROS and activated intracellular signaling pathways, mainly the p38/AKT signaling pathway. These results indicate that heat-inactivated FBS may regulate the p38/AKT signaling pathway by promoting the production of appropriate amounts of ROS, thereby regulating cell viability.