Gliomas, the most prevalent type of brain tumor in adults, are associated with high rates of morbidity and mortality. Recent studies on (131)I labeled scorpion toxins suggest they can be developed as tumor-specific agents for glioma diagnosis and treatment. This study investigated the potential of (131)I labeled Buthus martensii Karsch chlorotoxin ((131)I-BmK CT) as a new approach for targeted imaging and therapy of glioma. The results showed that (131)I can be successfully linked to BmK CT with satisfactory radiochemical purity and stability and that (131)I-BmK CT markedly inhibited glioma cell growth in a dose and time dependent manner, with significant accumulation in glioma cells in vitro. Persistent intratumoral radioiodine retention and specific accumulation of (131)I-BmK CT were observed in C6 glioma tumor, which was clearly visualized by SPECT imaging. Both intratumoral and intravenous injections of (131)I-BmK CT could result in significant tumor inhibition efficacy and prolonging the lifetime of tumor-bearing mice. Based on these promising results, it is concluded that (131)I-BmK CT has the potential to be explored as a novel tool for SPECT imaging and radionuclide therapy of glioma.