Metastatic melanoma cells overexpressing gap junctions were assayed for their ability to propagate cell death by a novel combination therapy that generates reactive oxygen species (ROS) by both 1) non-thermal plasma (NTP) and 2) tirapazamine (TPZ) under hypoxic conditions. Results demonstrate additive-to-synergistic effects of combination therapy compared to each agent individually. NTP induces highly localized cell death in target areas whereas TPZ partially reduces viability over the total surface area. However, when high gap junction expression was induced in melanoma cells, effects of combination NTP+TPZ therapy was augmented, spreading cell death across the entire plate. Similarly, in vivo studies of human metastatic melanoma in a mouse tumor model demonstrate that the combined effect of NTP+TPZ causes a 90% reduction in tumor volume, specifically in the model expressing gap junctions. Treatment with NTP+TPZ increases gene expression in the apoptotic pathway and oxidative stress while decreasing genes related to cell migration. Immune response was also elicited through differential regulation of cytokines and chemokines, suggesting potential for this therapy to induce a cytotoxic immune response with fewer side effects than current therapies. Interestingly, the gap junction protein, Cx26 was upregulated following treatment with NTP+TPZ and these gap junctions were shown to maintain functionality during the onset of treatment. Therefore, we propose that gap junctions both increase the efficacy of NTP+TPZ and perpetuate a positive feedback mechanism of gap junction expression and tumoricidal activity. Our unique approach to ROS induction in tumor cells with NTP+TPZ shows potential as a novel cancer treatment.