In this paper, 10-hydroxycamptothecin (HCPT)-loaded poly (n-butyl cyanoacrylate) nanoparticles (HCPT-PBCA-NPs) co-modified with polysorbate 80, soybean phospholipids, and polyethylene glycol (100) monostearate were successfully prepared via miniemulsion polymerization, and were characterized for particle size, morphology, zeta potential, encapsulation efficiency (EE) and drug loading capacity (DL). The chemical structure of HCPT-PBCA-NPs and the state of HCPT in the PBCA-NPs were investigated by DSC, FTIR and H NMR. Additionally, drug release, cytotoxicity, cellular uptake capacity, cellular uptake mechanism, and in vivo behavior of NPs were investigated as well. The particles were 92.7nm in size with a high EE of 94.24%. FTIR, H NMR, and DSC demonstrated complete polymerization of BCA monomers and the drug was in a molecular or amorphous form inside the NPs. In vitro release of the drug from HCPT-PBCA-NPs exhibited sustained-release and less than 60% of HCPT was released from the NPs within 24h of dialysis. Cellular uptake study displayed that Caco-2 cell uptake of NPs was governed by active endocytosis, clathrin- and caveolin-mediated process, and increased with the increase of the NPs concentration and the time. The pharmacokinetic study in rats showed that encapsulation of HCPT into PBCA-NPs increased the C and AUC about 6.52 and 7.56 times, respectively, in comparison with the HCPT suspension. It was concluded that HCPT loaded PBCA-NPs prepared by miniemulsion polymerization could be promising in oral drug delivery.