Sublethal doses of γ-rays promote cancer cell invasion by stimulating a signaling pathway that sequentially involves p53, sulfatase 2 (SULF2), β-catenin, interleukin-6 (IL-6), signal transducer and activator of transcription 3 (STAT3), and Bcl-XL. Given that Bcl-XL can increase O2•- production by stimulating respiratory complex I, the possible role of mitochondrial reactive oxygen species (ROS) in γ-irradiation-induced cell invasion was investigated. Indeed, γ-irradiation promoted cell invasion by increasing mitochondrial ROS levels, which was prevented by metformin, an inhibitor of complex I. γ-Irradiation-stimulated STAT3 increased the expression of superoxide dismutase 2 (SOD2), a mitochondrial enzyme that catalyzes the conversion of O2•- to hydrogen peroxide (H2O2). In contrast to O2•-, H2O2 functions as a signaling molecule. γ-Irradiation consistently stimulated the Src-dependent invasion pathway in a manner dependent on both complex I and SOD2. SOD2 was also essential for the invasion of un-irradiated cancer cells induced by upregulation of Bcl-XL, an intracellular oncogene, or extracellular factors, such as SULF2 and IL-6. Overall, these data suggested that SOD2 is critical for the malignant effects of radiotherapy and tumor progression through diverse endogenous factors.