Surface charge plays an important role in determining the interactions of nanoparticles with biological components. Substantial studies have demonstrated that surface charge affects the fate of nanoparticles after intravenous administration; however, few reports investigate the effect of surface charge on the bioavailability and absorption of nanoparticles after oral administration. In this study, polymeric nanoparticles with similar particle size and surface poly(ethylene glycol) (PEG) density, whereas with varying surface charges (positive, negative and neutral), were developed to study the effect of surface charge on oral absorption of polymeric nanoparticles. The nanoparticles were constructed from poly(ethylene glycol)-block-polylactic acid (PEG-PLA) with the incorporation of lipid components with different charges. Our results suggested that positive surface charge facilitated the cellular uptake and transport of nanoparticles through both Caco-2 cells in vitro and small intestinal epithelial cells in vivo. The positively charged nanoparticles showed a favorable distribution in the small intestine, and significantly improved oral bioavailability. This study presents valuable information towards the design of nanoparticles for improved oral drug delivery.