All-trans-retinoic acid (RA) is a non-toxic physiological metabolite of vitamin A. A paclitaxel (PTX) prodrug (RA-PTX) with high PTX content of 75% was synthesized via an easy condensation reaction. RA-PTX nanoparticles (RA-PTX NPs) were prepared through a nanoprecipitation method which increased the water solubility of PTX. RA-PTX NPs were spherical in shape and exhibited favorable structural stability in both water and the physiological environment. RA-PTX NPs possessed effective cellular uptake as revealed by confocal laser scanning microscopy and exerted potent cytotoxicity. These results highlight the potential of nanomedicines from PTX prodrugs for increasing the drug loading and water solubility of PTX.