The purpose of this study was to characterize the properties in vitro, i.e. release,degradation, hemolytic potential and anticancer activity, and in vivo disposition ofall-trans-retinoic acid (ATRA) in rats after administration of ATRA-loaded micelle-likenanoparticles. The amphiphilic block copolymers consisted of a micellar shell-formingmPEG block and a core-forming PLA block. The mPEG–PLA nanoparticles prepared byan acetone volatilization dialysis procedure were identified as having core–shell structure by1H NMR spectroscopy. Critical association concentration, drug contents, loading efficiency, particle sizeand ξ potential were evaluated. The release of ATRA from the nanoparticles and thedegradation of PLA were found to be mostly associated with the compositions of thenanoparticles. ATRA release was faster at smaller molecular weight of copolymerand lower drug contents. In vitro, the incorporation of ATRA in mPEG–PLAnanoparticles reduced the hemolytic potential of ATRA. Furthermore, anticancer activityof ATRA against HepG2 cell was increased by encapsulation, which showed anenhancement of tumor treatment of ATRA. In vivo, after intravenous injection torats, the levels of ATRA in the blood stream and the bioavailability were higherfor ATRA-loaded mPEG–PLA nanoparticles than those for ATRA solution. Inconclusion, the structure of the mPEG–PLA diblock copolymer could be modulatedto fit the demand of in vitro and in vivo characterizations of nanoparticles. ThemPEG–PLA nanoparticles’ loading ATRA have a promising future for injectionadministration.