A major goal of breast cancer research is to prevent the molecular events that lead to tumour metastasis. It is well-established that both cytoplasmic and mitochondrial reactive oxygen species (ROS) play important roles in cell migration and metastasis. Accordingly, this study examined the molecular mechanisms of the anti-metastatic effects of NecroX-5, a mitochondrial ROS scavenger. NecroX-5 inhibited lung cancer metastasis by ameliorating migration in a mouse model. In human cancer cells, the inhibition of migration by NecroX-5 is cell type-dependent. We observed that the effect of NecroX-5 correlated with a reduction in mitochondrial ROS, but mitochondrial ROS reduction by MitoQ did not inhibit cell migration. NecroX-5 decreased intracellular calcium concentration by blocking Ca2+ influx, which mediated the inhibition of cell migration, AKT downregulation and the reduction of mitochondrial ROS levels. However, the reduction of mitochondrial ROS was not associated with supressed migration and AKT downregulation. Our study demonstrates the potential of NecroX-5 as an inhibitor of breast cancer metastasis.