Abstract We prepared methotrexate (MTX)-encapsulated polymeric nanoparticles using methoxy poly(ethylene glycol) (MPEG)-grafted chitosan (ChitoPEG) copolymer. MTX-encapsulated polymeric nanoparticles of ChitoPEG copolymer has around 50–300 nm in particle size and showed spherical shape when observed by transmission electron microscope (TEM). In 1H nuclear magnetic resonance (NMR) study, the specific peaks of MTX and chitosan as a drug carrying inner-core disappeared at D 2O and only the specific peak of MPEG was observed, while specific peaks of MPEG, MTX, and chitosan appeared in DCl/D 2O mixtures. These results indicated that MTX was complexed with chitosan and then core–shell type nanoparticles had formed in aqueous environment, i.e., MTX/chitosan complexes composed of inner-core and MPEG composed of outer-shell of the nanoparticles. Loading efficiency of MTX in the polymeric nanoparticles was 94% (w/w) of ChitoPEG-1, 91.1% (w/w) of ChitoPEG-2, 90.1% (w/w) of ChitoPEG-3 and 65.2% (w/w) of ChitoPEG-4, expectively. The higher the drug feeding ratio, the higher the drug content and the lower the loading efficiency. The higher the MPEG graft ratio in the copolymer, the lower the drug content and loading efficiency. Drug contents evaluated by 1H NMR were the same as found by UV spectrophotometer.