A novel rhein formulation based on poly(lactic- co-glycolic acid) (PLGA) nanoparticles (NPs) suitable for oral administration was developed in this study. The designed nanosystems were obtained by a modified spontaneous emulsification solvent diffusion method. The morphology of rhein-loaded PLGA NPs showed a spherical shape with a smooth surface, without any particle aggregation. Mean size of the NPs was 140.5±4.3 nm, and the zeta potential was −16.9±3.1 mV. The average drug loading was 3.9%±0.7%, and encapsulation efficiency was 84.5%±6.2%. Meanwhile, NPs are characterized by the slower release (only about 70% of rhein is released within 5 hours), and the model that fitted best for rhein released from the NPs was Higuchi kinetic model with correlation coefficient r=0.9993, revealing that rhein could be controlled released from the NPs. In vivo, NPs altered the distribution of rhein, and the half-life after oral administration was prolonged remarkably more than those of suspensions (22.6 hours vs 4.3 hours). The pharmacokinetic results indicated that the NPs had sustained-release efficacy. The area under the curve 0–∞ of the NPs formulation was 3.07-fold higher than that of suspensions, suggesting that the encapsulated rhein had almost been absorbed in rats over the period of 12 hours. Although rhein-loaded PLGA NP formulations are hopefully used as a chemotherapeutic or adjuvant agent for human gastric cancer (SGC-7901), their in vivo antitumor effect and mechanisms at the molecular level still need further study.