Two types of 10-hydroxycamptothecin (10-HCPT) loaded nanostructured lipid carriers (NLC-F 68 and NLC-Brij 35) intended for use as the alternative formulation of 10-HCPT for parenteral administration were prepared using an emulsification-ultrasonication method and fully characterized from physicochemical and in vitro release standpoint. The particle size was measured by laser diffraction, being 108 nm and 126 nm for NLC-F 68 and NLC-Brij 35, respectively. Zeta potentials of two NLCs were -28.5 mV and -32.1 mV analyzed by photon correlation spectroscopy. The incorporated efficiency was more than 85%. It is observed that NLCs are homogeneous and spherical in shape by transmission electron microscopy. Differential scanning calorimetry analysis of NLCs showed that 10-HCPT was dispersed within NLC in an amorphous state. The combination of trehalose and mannitol as cryoprotectant was most suitable for HCPT-NLC lyophilization. The in vitro release behavior for two types of NLC was similar and displayed biphasic drug release pattern with rapid release at the initial stage and prolonged release afterwards. These results suggest that NLC could be exploited as a carrier of 10-HCPT with high incorporation efficiency and controlled release and that NLC may serve as the alternative delivery system for parenteral administration of 10-HCPT.