Tumor-penetrating peptides (TPPs) have been discovered and well recognized for their unique tumor-penetrating properties, which allows for the selective delivery of therapeutic agents and diagnostics to tumor sites and mediates their distribution deep in the tumor parenchyma. The mechanisms that such peptides take advantage of to gain access to tumor parenchyma through the C-end Rule or CendR pathway after second sequence motif is exposed at the C-terminus of the peptide have been intensively investigated. Although the pathological function of the CendR pathway still remains unclear, the endocytosis uptake based on this route has shown very distinctive features in terms of tumor penetration and drug delivery. This chapter focuses on the discovery, molecular structure, drug delivery, and molecular imaging applications of TPPs. The internalization mechanisms with regards to sequence and structure of tumor penetrating peptides are also briefly summarized.