Nanomedicines can effectively penetrate tumor sites compared to traditionally used drugs. However, effective drugs that reach the interior of tumors remain limited. Based on studies of the complex tumor microenvironment, we summarized the barriers restricting tumor penetration of nanomedicines in this review. Penetration barriers are mainly caused by tumor blood vessels, stroma, and cell abnormalities. The repair of abnormal tumor blood vessels and tumor stroma and adjusting the physicochemical properties of nanoparticles are considered promising strategies to improve the tumor permeation of nanomedicines. The effects of nanoparticle properties, including size, shape, and surface charge, on tumor penetration were also reviewed. We expect to provide research ideas and a scientific basis for nanomedicines to increase intratumoral permeability and improve anti-tumor effects.