Biological barriers are essential physiological protective systems and obstacles to drug delivery. Nanoparticles (NPs) can access the paracellular route of biological barriers, either causing adverse health impacts on humans or producing therapeutic opportunities. This Review introduces the structural and functional influences of NPs on the key components that govern the paracellular route, mainly tight junctions, adherens junctions, and cytoskeletons. Furthermore, we evaluate their interaction mechanisms and address the influencing factors that determine the ability of NPs to open the paracellular route, which provides a better knowledge of how NPs can open the paracellular route in a safer and more controllable way. Finally, we summarize limitations in the research models and methodologies of the existing research in the field and provide future research direction. This Review demonstrates the in-depth causes for the reversible opening or destruction of the integrity of barriers generated by NPs; more importantly, it contributes insights into the design of NP-based medications to boost paracellular drug delivery efficiency.