Cold atmospheric plasma (CAP) has been proposed as a novel promising anti-cancer treatment modality. Apoptosis and necrosis have been revealed in CAP-induced cell death, but whether CAP induces pyroptosis, another kind of programmed cell death is still unknown. In the present study, we first reported that CAP effectively induced pyroptosis in a dose-dependent manner in Gasdermin E (GSDME) high-expressed tumor cell lines. Interestingly, the basal level of GSDME protein was positively correlated with the sensitivity to CAP in three selected cancer cell lines, implying GSDME might be a potential biomarker of prognosis in the forthcoming cancer CAP treatment. Moreover, our study revealed that CAP-induced pyroptosis depended on the activation of mitochondrial pathways (JNK/cytochrome c/caspase-9/caspase-3) and the cleavage of GSDME but not Gasdermin D (GSDMD). ROS generation induced by CAP was identified to initiate the pyroptotic signaling. These results complemented our knowledge on CAP-induced cell death and provide a strategy to optimize the effect of CAP cancer treatment.