There is growing evidence that plasma-activated medium (PAM), which is prepared by non-thermal plasma (NTP) irradiation of cell-free medium, is a beneficial tool for cancer therapy. PAM has been reported to preferentially kill cancer cells; however, its mechanism is not fully understood. Since PAM contains reactive oxygen species (ROS) and reactive nitrogen species, the anti-cancer effects of PAM are thought to be attributed to oxidative stress induced by these reactive molecules. Oxidative stress has been shown to release zinc (Zn2+) from intracellular Zn2+ stores and provoke Zn2+-dependent cell death. We have previously demonstrated that intracellular free Zn2+ plays a critical role in PAM-induced cell death in human neuroblastoma SH-SY5Y cells. In this study, we found that normal human fibroblasts were less susceptible to PAM cytotoxicity compared with SH-SY5Y cells. PAM decreased intracellular NAD(+) levels in both cells, whereas the depletion of ATP and mitochondrial ROS generation was hardly observed in fibroblasts. Intracellular mobile Zn2+ contents of fibroblasts were lower than those of SH-SY5Y cells. PAM suppressed the activity of aconitase, which is a tricarboxylic acid cycle enzyme, only in SH-SY5Y cells, and N',N',N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn2+ chelator, counteracted the suppression. The combination treatment with PAM and Zn2+ augmented PAM-induced ATP depletion, mitochondrial ROS generation, and cytotoxicity in fibroblasts. These findings suggest the possibility that cells with high intracellular mobile Zn2+ are susceptible to PAM cytotoxicity. Therefore, we concluded that the differences in mobile Zn2+ levels affect PAM-induced cellular responses.