SN-38 is a highly effective drug against many cancers. The development of an optimal delivery system for SN-38 is extremely challenging due to its low solubility and labile lactone ring. Herein, SN-38 encapsulated in poly(D,L-lactide-co-glycolide) nanoparticles (NPs) is introduced to enhance its solubility, stability and cellular uptake. SN-38-loaded NPs prepared by spontaneous emulsification solvent diffusion (SESD) method had an average diameter of 310 nm, a zeta potential of -9.69 mV and a loading efficiency of 71%. They were able to protect the active lactone ring of SN-38 against inactivation under physiological condition. A colorectal adenocarcinoma cell line (COLO-205) was used to assess the NPs effects on cytotoxicity and cellular uptake. Result showed a significant decreased cell proliferation and cell apoptosis. These results suggest that these SN-38-loaded NPs can be an effective delivery system for the treatment of colon cancer and potentially for other types of cancers.