Peptide amphiphiles (PAs) are a class of molecules that combine the structural features of amphiphilic surfactants with the functions of bioactive peptides. They self-assemble into one-dimensional (1D) nanostructures, primarily nanofibers with a cylindrical geometry, which are highly bioactive and have applications in tissue engineering, regenerative medicine, and drug delivery. The authors highlight their strategies for using molecular self-assembly to produce PAs and their translation into therapeutic applications. They discuss the design and assembly mechanisms of PAs, including the role of hydrophobic interactions, hydrogen bonding, and electrostatic repulsions. The internal structures of PA nanofibers and their control through molecular design, assembly environment, and co-assembling molecules are also explored. The applications of PA nanofibers, such as encapsulation of small molecules, cross-linking chemistry, mineralization templating, magnetic resonance imaging (MRI), light-activated self-assembly, surface patterning, and their use in regenerative medicine, including neural regeneration, hard tissue replacement, and angiogenesis, are detailed. The potential of PAs in treating spinal cord injury, inducing angiogenesis, and regenerating hard tissues is emphasized.Peptide amphiphiles (PAs) are a class of molecules that combine the structural features of amphiphilic surfactants with the functions of bioactive peptides. They self-assemble into one-dimensional (1D) nanostructures, primarily nanofibers with a cylindrical geometry, which are highly bioactive and have applications in tissue engineering, regenerative medicine, and drug delivery. The authors highlight their strategies for using molecular self-assembly to produce PAs and their translation into therapeutic applications. They discuss the design and assembly mechanisms of PAs, including the role of hydrophobic interactions, hydrogen bonding, and electrostatic repulsions. The internal structures of PA nanofibers and their control through molecular design, assembly environment, and co-assembling molecules are also explored. The applications of PA nanofibers, such as encapsulation of small molecules, cross-linking chemistry, mineralization templating, magnetic resonance imaging (MRI), light-activated self-assembly, surface patterning, and their use in regenerative medicine, including neural regeneration, hard tissue replacement, and angiogenesis, are detailed. The potential of PAs in treating spinal cord injury, inducing angiogenesis, and regenerating hard tissues is emphasized.