Self-assembling peptides are biomedical materials with unique structures that are formed in response to various environmental conditions. Governed by their physicochemical characteristics, the peptides can form a variety of structures with greater reactivity than conventional non-biological materials. The structural divergence of self-assembling peptides allows for various functional possibilities; when assembled, they can be used as scaffolds for cell and tissue regeneration, and vehicles for drug delivery, conferring controlled release, stability, and targeting, and avoiding side effects of drugs. These peptides can also be used as drugs themselves. In this review, we describe the basic structure and characteristics of self-assembling peptides and the various factors that affect the formation of peptide-based structures. We also summarize the applications of self-assembling peptides in the treatment of various diseases, including cancer. Furthermore, the in-cell self-assembly of peptides, termed reverse self-assembly, is discussed as a novel paradigm for self-assembling peptide-based nanovehicles and nanomedicines.