Previous studies have revealed that lactate dehydrogenase A (LDHA) exhibited an indirect antioxidative activity, which played a crucial role in preventing cancer cells from oxidative stress. Here we demonstrated that, apart from antioxidative activities, LDHA and LDHB displayed prooxidative activity in cancer cells. In aqueous phase, LDHA and B both exhibited ROS-generating activity and LDHB was more active than LDHA. In cancer cells, the dominant antioxidative activity of LDH can be switched to dominant prooxidative activity or vice versa, depending on the strength of oxidative stimuli, indicating that LDH were bifunctional. Moreover, we demonstrated that mitochondrial superoxide served as an initiator to trigger LDH-catalyzed amplification of ROS. The oxidative stimuli, such as modulators of electron transfer chain and anticancer agents that kill cancer cells via ROS induction, induced a ROS generation involving 2 phases, induction of mitochondrial superoxide and amplification of ROS by LDH.