Biodegradable polyester nanoparticles have now attracted growing interest as promising drug delivery system. However, a fundamental understanding about its cellular transport as well as the influence by the polymeric architecture is still lack, which remains a significant obstacle to optimal nanocarrier design. In this work, using Caco-2 cell model, we characterized the cellular transport pathway of pegylated polyester nanoparticles and determined the effect of polymer architecture including PEG chain length and core material on its cellular interaction and transcellular transport. The nanoparticles were found to undergo an energy-dependent, lipid raft-mediated, but caveolae-independent endocytosis. PEG chain length (from 2000 to 5000 Da) and core material (PLA/PLGA) hardly affected the cellular interaction and the intracellular itinerary of the nanoparticles. However, in the case of transcellular transport, the maximal transcellular transport efficiency for its payload was achieved by the PEG5000-PLA40000 nanoparticles which present higher drug loading capacity and slower drug release. The findings here revealed the cellular interaction mechanism of pegylated polyester nanoparticles and provided evidence for the role of polymer architectures in modulating the transcellular permeability of the agents loaded by the nanoparticles, and would be helpful in improving carrier design to enhance drug delivery.