Nanoparticles (NPs) have emerged as an effective modality for the treatment of various diseases including cancer, cardiovascular and inflammatory diseases. Various forms of NPs including liposomes, polymer particles, micelles, dendrimers, quantum dots, gold NPs and carbon nanotubes have been synthesized and tested for therapeutic applications. One of the greatest challenges that limit the success of NPs is their ability to reach the therapeutic site at necessary doses while minimizing accumulation at undesired sites. The biodistribution of NPs is determined by body's biological barriers that manifest in several distinct ways. For intravascular delivery of NPs, the barrier manifests in the form of: (i) immune clearance in the liver and spleen, (ii) permeation across the endothelium into target tissues, (iii) penetration through the tissue interstitium, (iv) endocytosis in target cells, (v) diffusion through cytoplasm and (vi) eventually entry into the nucleus, if required. Certain applications of NPs also rely on delivery through alternate routes including skin and mucosal membranes of the nose, lungs, intestine and vagina. In these cases, the diffusive resistance of these tissues poses a significant barrier to delivery. This review focuses on the current understanding of penetration of NPs through biological barriers. Emphasis is placed on transport barriers and not immunological barriers. The review also discusses design strategies for overcoming the barrier properties.