It is known that artesunate (ART) induced apoptosis is due to the reactive oxygen species (ROS) generation which triggers many apoptosis. DCFH-DA and Rhodamine 123 were used to probe the level of ROS and mitochondrial membrane potential. Time-lapse confocal fluorescence microscopy was used to monitor the dynamics of ROS generation and the loss of mitochondrial membrane potential during ART-induced human lung adenocarcinoma cells (ASTC-a-1) apoptosis. The data show that the cell ability can be reduced by ART of 0~50 μg/mL. ART of 40 μg/mL which be used to generate significantly ROS can induce notablely loss of mitochondrial membrane potential. Results show that N-acetylcysteine (NAC), a scavenger of ROS, can significantly inhibits the ART-induced apoptosis and the loss of mitochondrial membrane potential and ART induces ROS-mediated apoptosis and loss of mitochondrial membrane potential.