Voltage-dependent anion channels (VDACs) are key proteins involved in regulating mitochondrial membrane permeability and cellular stress responses. In this study, we identified and characterized nine MeVDAC genes in cassava ( Manihot esculenta) through bioinformatic analysis of the whole cassava genome. These genes were found to contain conserved Porin_3 domains, typical of mitochondrial pore proteins, and exhibited diverse expression patterns across various tissues, particularly in root tips, suggesting their roles in growth, development, and stress response. Cis-acting element analysis revealed that MeVDAC genes are involved in multiple stress signaling pathways, including those related to drought, salt, cold, and oxidative stress. Among these, MeVDAC2 was selected for further investigation due to its unique stress-related cis-elements and lack of replication events. Overexpression of MeVDAC2 in both yeast and cassava significantly enhanced tolerance to sodium, cadmium, and lead toxicities. In addition, MeVDAC2-overexpressing plants showed reduced reactive oxygen species (ROS) accumulation, lower malondialdehyde (MDA) levels, and increased antioxidant enzyme activities under stress conditions, indicating improved oxidative stress tolerance. Our findings suggest that MeVDAC2 plays a crucial role in mitigating metal ion toxicity and oxidative stress in cassava, and its overexpression could be utilized to enhance cassava’s resilience to abiotic stresses. This study provides new insights into the functional diversity of VDACs in plants and highlights their potential for improving crop stress tolerance.