The fate of intravenously injected nanoparticles (NPs) is significantly affected by nano-protein interaction and corona formation. However, such an interaction between NPs and digestive enzymes occurring in the gastrointestinal tract (GIT) and its impacts on epithelial cell uptake are little known. We synthesized the poly(3-hydroxybutyrate- co-3-hydroxyhexanoate)-based cationic NPs (CNPs) and investigated the CNP-digestive enzyme interaction and its effect on the cellular uptake. The formation of enzyme corona was confirmed by size/zeta potential analysis, morphology, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and enzyme quantification. The cellular uptake of CNPs by Caco-2 cells was significantly reduced upon the formation of enzyme corona. Our findings demonstrate the digestive enzyme corona formation and its inhibited effect on the epithelial cell uptake of CNPs for the first time. Understanding the enzyme corona could offer a new insight into the fate of nanomedicines in the GIT, and this understanding would be highly beneficial for guiding future nanomedicine designs.