Aim: To determine whether glioma cells can be specifically and efficiently targeted by superparamagnetic iron oxide nanoparticle (SPIO)-fluorescein isothiocyanate (FITC)-chlorotoxin (SPIOFC) that is detectable by magnetic resonance imaging (MRI) and optical imaging. Methods: SPIOFC was synthesized by conjugating SPIO with FITC and chlorotoxin. Glioma cells (human U251-MG and rat C6) were cultured with SPIOFC and SPIOF (SPIO-FITC), respectively. Neural cells were treated with SPIOFC as the control for SPIOFC-targeted glioma cells. The internalization of SPIOFC by glioma cells was assessed by MRI and was quantified using inductively-coupled plasma emission spectroscopy. The optical imaging ability of SPIOFC was evaluated by confocal laser scanning microscopy. Results: Iron per cell of U251 (72.5±1.8 pg) and C6 (74.9±2.2 pg) cells cultured with SPIOFC were significantly more than those of U251 (6.6±1.0 pg) and C6 (7.1±0.8 pg) cells incubated with SPIOF. The T2 signal intensity of U251 and C6 cells cultured with SPIOFC (233.6±25.9 and 211.4±17.2, respectively) were substantially lower than those of U251 and C6 cells incubated with SPIOF (2275.3±268.6 and 2342.7±222.4, respectively). Moreover, there were significant differences in iron per cell and T2 signal intensity between SPIOFC-treated neural cells (1.3±0.3; 2533.6±199.2) and SPIOFC-treated glioma cells. SPIOFC internalized by glioma cells exhibited green fluorescence by confocal laser scanning microscopy. Conclusion: SPIOFC is suitable for the specific and efficient targeting of glioma cells. MRI and optical imaging in conjunction with SPIOFC can differentiate glioma cells from normal brain tissue cells.