Because mitochondria are essential organelles for regulating energy homeostasis and intrinsic apoptosis, the perturbation of mitochondrial functions has been considered as an anticancer treatment. In this study, a new near-infrared (NIR) fluorescent probe, SiR-Mito11 was developed as a theragnostic agent for brain tumor by targeting mitochondria. SiR-Mito11 exhibited potential anticancer activity against glioma cells but tolerance in normal neuronal cells. We further confirmed that the selective accumulation of SiR-Mito11 in glioma cells disrupted mitochondria membrane potential, followed by apoptotic cell death.