Biological barriers protect delicate internal tissues from exposures to and interactions with hazardous materials. Primary anatomical barriers prevent external agents from reaching systemic circulation and include the pulmonary, gastrointestinal, and dermal barriers. Secondary barriers include the blood–brain, blood–testis, and placental barriers. The tissues protected by secondary barriers are particularly sensitive to agents in systemic circulation. Neurons of the brain cannot regenerate and therefore must have limited interaction with cytotoxic agents. In the testis, the delicate process of spermatogenesis requires a specific milieu distinct from the blood. The placenta protects the developing fetus from compounds in the maternal circulation that would impair limb or organ development. Many biological barriers are semi-permeable, allowing only materials or chemicals, with a specific set of properties, that easily pass through or between cells. Nanoparticles (particles less than 100 nm) have recently drawn specific concern due to the possibility of biological barrier translocation and contact with distal tissues. Current evidence suggests that nanoparticles translocate across both primary and secondary barriers. It is known that the physicochemical properties of nanoparticles can affect biological interactions, and it has been shown that nanoparticles can breach primary and some secondary barriers. However, the mechanism by which nanoparticles cross biological barriers has yet to be determined. Therefore, the purpose of this review is to summarize how different nanoparticle physicochemical properties interact with biological barriers and barrier products to govern translocation.