Gliomas, the most prevalent type of brain tumor in adults, are associated with high rates of morbidity and mortality. Recent studies on I labeled scorpion toxins suggest they can be developed as tumor-specific agents for glioma diagnosis and treatment. This study investigated the potential of I labeled Buthus martensii Karsch chlorotoxin (I-BmK CT) as a new approach for targeted imaging and therapy of glioma. The results showed that I can be successfully linked to BmK CT with satisfactory radiochemical purity and stability and that 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 I-BmK CT were observed in C6 glioma tumor, which was clearly visualized by SPECT imaging. Both intratumoral and intravenous injections of 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 I-BmK CT has the potential to be explored as a novel tool for SPECT imaging and radionuclide therapy of glioma.