Previous studies have demonstratedf that heat stress can induce injury of the central nervous system and lead to neuronal cell apoptosis. However, the molecular mechanisms underlying these cellular changes remain unclear. In the present study, flow cytometry was used to investigate heat-stress-induced apoptosis, and caspase-3 activation was also assessed in neurons. The role of reactive oxygen species (ROS) accumulation in the heat-stress-induced apoptosis of neurons was demonstrated using the antioxidant drug manganese (III) tetrakis (4-benzoic acid)porphyrin. The present study presents evidence that heat stress induces mitogen-activated protein kinase (MAPK) activation in rat malignant glioma F98 cells. Following the inhibition of different MAPKs with a range of specific inhibitors, SB203580 (an inhibitor of p38 MAPK), but not PD98059 (an inhibitor of extracellular signal-regulated kinases) or SP600125 (an inhibitor of c-Jun N-terminal kinases), diminished the production of ROS and apoptosis, and prevented activation of the p38-downstream kinase MAPK-activated protein kinase 2 (MK2) in neurons. Inhibiting MK2 with dominant negative adenoviral constructs or a specific inhibitor significantly decreased normal and heat-stress-induced ROS accumulation and cell apoptosis, whereas inhibition of another kinase downstream of p38 MAPK, MAPK-activated protein kinase 5, by transfection with another adenoviral construct did not exert the same effects. Taken together, these findings indicate that heat stress stimulation induces p38-MK2 pathway activation, which exerts a pro-apoptotic effect by regulating ROS accumulation in neurons.