Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. Although increasing evidence suggests that NTP selectively induces apoptosis in some types of tumor cells, the molecular mechanisms underlying this phenomenon remain unclear. In this study, we further investigated possible molecular mechanisms for NTP-induced apoptosis of HeLa cells. The results showed that NW exposure significantly inhibited the growth and viability of HeLa cells. Morphological observation and flow cytometry analysis demonstrated that NW exposure induced HeLa cell apoptosis. NTP exposure also activated caspase-9 and caspase-3, which subsequently cleaved poly (ADP-ribose) polymerase. Furthermore, NTP exposure suppressed Bcl-2 expression, enhanced Bax expression and trans location to mitochondria, activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c. Further studies showed that NTP treatment led to ROS generation, whereas blockade of ROS generation by N-acetyl-L-cysteine (NAC, ROS scavengers) significantly prevented NTP-induced mitochondrial alteration and subsequent apoptosis of HeLa cells via suppressing Bax translocation, cytochrome c and caspase-3 activation. Taken together, our results indicated that NW exposure induced mitochondria-mediated intrinsic apoptosis of HeLa cells was activated by ROS generation. These findings provide insights to the therapeutic potential and clinical research of NW as a novel tool in cervical cancer treatment. (C) 2017 Published by Elsevier Inc.