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Development of nanoparticle (NP) to simultaneously overcome the diffusion and absorption barrier has drawn much attention. Our group recently demonstrated that the virus mimicking dilauroylphosphatidylcholine functionalized NP (DLPC-NP) could rapidly penetrate across mucus layer and subsequently enter epithelia effectively, and exhibited high potential for oral drug delivery. However, the safety of the NP has provoked some scientific concerns, which debates from their properties that circuiting the protections of mucus and exhibiting strong interaction with cell membrane. To be further developed for oral drug delivery, the possible toxicity of DLPC-NPs was systematically evaluated in current study. For in vitro studies, the exposure of NPs to Caco-2 cells had no effect on cell viability and membrane integrity. Then the oxidative stress, cytokine production and genotoxicity assay indicated that the NPs did not induce any cell responses. The hemolysis test also demonstrated the good hemocompatibility of NPs. For in vivo studies, the biodistribution results showed that a large proportion of DLPC-NPs were detected in liver (0.51 O.D%/g), and followed by kidney (0.39 O.D%/g). Then the hematology test, blood biochemical assay and pathological assay were performed after a long oral administration periods of 28 days. All the results indicated that the NPs could not induce any toxic response or histopathological lesions. Thus, based on these results, it can be concluded that the zwitterion-functionalized NPs might be a biocompatible potential candidate for oral drug delivery. More importantly, we aimed to emphasis the safety of the nanocarriers and also provide a reference for toxicity studies.