T-2 toxin at low concentrations can induce ROS accumulation and modulate host resistance in plants. NOX plays crucial roles in ROS production and is regulated by Ca²⁺via direct binding to EF-hand motifs. In this study, the effect of EGTA (Ca²⁺ chelating agent) on the expression and enzymatic activity of NOX, as well as the activities and corresponding gene expressions involved in ROS metabolism and cell membrane integrity, were investigated in treated slices. Results indicated that EGTA treatment significantly affected gene expression and activity of NOX, and reduced ROS accumulation and cell membrane integrity and the enzymatic activities and gene expression involved in ROS metabolism when exposed to treatment. The addition of exogenous Ca²⁺ restored the initial relative transcript abundance, ROS accumulation and their activities. Results suggest that Ca²⁺ affected by EGTA plays a crucial role in NOX activity regulation, ultimately affecting ROS metabolism in slices induced by T-2 toxin.