Self-assembled nanoparticles of cholesterol-modified O-carboxymethyl chitosan (CCMC) were prepared to be used as a novel carrier for paclitaxel (PTX) in this study. CCMC-6.9 was synthesized by the covalent conjugation of cholesterol to O-carboxymethyl chitosan with the succinyl linkage and the degree of substitution (DS) of the cholesterol moiety was 6.9%. CCMC-6.9 formed self-assembled nanoparticles with a size of 209.5 nm in aqueous media. Paclitaxel-loaded CCMC-6.9 self-assembled nanoparticles were prepared using a dialysis method and their characteristics were analyzed by dynamic laser light scattering (LLS), transmission electron microscopy (TEM) and ultraviolet spectroscopy (UV). PTX-loaded CCMC-6.9 self-assembled nanoparticles were almost spherical in shape and their size increased from 245.6 to 355.3 nm with PTX-loading content increasing from 18.7% to 34.9%. In vitro release of PTX from CCMC-6.9 self-assembled nanoparticles was carried out by the dynamic dialysis method. PTX continuously released in phosphate buffered saline (PBS) solutions for 84 h at 37 °C and its release was sensitive to the pH of the release media. The biodistribution of PTX-loaded CCMC-6.9 self-assembled nanoparticles was studied in female Balb/c mice. Compared with PTX in the solution of Cremophor EL (polyethoxylated castor oil)/ethanol (PTX-Cre), CCMC-6.9 self-assembled nanoparticles significantly increased the uptake of PTX in plasma, liver and spleen, but decreased the uptake in heart and kidney. These results suggest that CCMC-6.9 self-assembled nanoparticles can effectively solubilize PTX and modify its tissue biodistribution, which may be advantageous in enhancing the therapeutic index and reducing the toxicity of PTX.