Esophageal cancer is a highly aggressive malignancy with a low response to standard anti-cancer therapies. There is an unmet need to develop new therapeutic strategies to improve the clinical outcomes of current treatments. Cold atmospheric plasma (CAP) is a promising approach for cancer treatment, and has displayed anticancer efficacy in multiple preclinical models. Recent studies have shown that the efficacy of CAP is positively correlated with intracellular reactive oxygen species (ROS) levels. This suggests that aggressively increasing intracellular ROS levels has the potential to further improve CAP-mediated anticancer efficacy. Glutamine plays an important role in cellular ROS scavenging after being converted to glutathione (GSH, a well-described antioxidant) under physiological conditions, so reducing intracellular glutamine levels seems to be a promising strategy. To test this hypothesis, we treated esophageal cancer cells with CAP while controlling the supply of glutamine. The results showed that glutamine did affect the anticancer effect of CAP, and the combination of CAP stimulation and glutamine deprivation significantly inhibited the proliferation of esophageal cancer cells compared to the control group (p < 0.05). Furthermore, flow cytometric analysis documented a significant increase in more than 10% in apoptosis and necrosis of esophageal cancer cells after this synergistic treatment compared to the control group (p < 0.05). Thus, these results provide the first direct evidence that the biological function of CAP can be modulated by glutamine levels and that combined CAP stimulation and glutamine deprivation represent a promising strategy for the future treatment of esophageal cancer.