UNLABELLED: Despite advances in polymeric nanoparticles (NPs) as effective delivery systems for anticancer drugs, rapid clearance from blood and poor penetration capacity in heterogeneous tumors still remain to be addressed. Here, a dual coating of poly (ethylene glycol)-poly (d,l-lactic acid) (PEG-PDLLA) and water-soluble chitosan oligosaccharide (CO) was used to develop PLGA-based NPs (PCPNPs) with colloidal stability for delivery of paclitaxel (PTX). The PCPNPs were prepared by a modified nanoprecipitation process and exhibited homogeneous size of 165.5nm, and slight positive charge (+3.54mV). The single PEG-PDLLA-coated PLGA NPs (PPNPs) with negative charge (-13.42mV) were prepared as control. Human breast cancer MDA-MB-231 cell and mice MDA-MB-231 xenograft model were used for in vitro and in vivo evaluation. Compared to Taxol®, both PCPNPs and PPNPs increased the intracellular uptake and exerted stronger inhibitory effect on tumor cells in vitro, especially for PCPNPs. Particularly, due to the near neutral surface charge and shielding by the dual coating, the blank cationic NP presented low cytotoxicity. With the synergistic action of PEG-PDLLA and CO, PCPNPs not only strongly inhibited macrophage uptake and extended the blood circulation time, but also improved the selective accumulation and interstitial penetration capacity to/in tumor site. Consequently, a significantly enhanced antitumor efficacy was observed for the cationic PCPNPs. Our findings suggest that, the dual PEG-PDLLA/CO coating can effective improve the tumor accumulation and interstitial penetration of NPs and, therefore may have great potential for tumor treatment. STATEMENT OF SIGNIFICANCE: Rapid clearance from blood and poor penetration capacity in heterogeneous tumors represent great challenge for polymeric nanoparticles (NPs) as effective delivery systems for anticancer drugs. This study provides a promising cationic nanoparticle (PCPNPs) with dual coating of chitosan oligosaccharide (CO) and PEG-PDLLA to address the above problem. The PCPNPs prepared with 165.5nm and slight positive charge (+3.54mV) showed an improved accumulation and interstitial penetration capacity to/in tumor site, and thus led to an enhanced antitumor efficacy. This is the first time to report the cooperative effect of PEG-PDLLA and CO on PLGA NPs in this field. This work can arouse broad interests among researchers in the fields of nanomedicine, nanotechnology, and drug delivery system.