Although self-assembled peptide nanostructures (SPNs) have shown potential as promising biomaterials, there is a potential problem associated with the extremely slow hydrolysis rate of amide bonds. Here, we report the development of cell-penetrating cross-beta SPNs with a controllable biodegradation rate. The designed self-assembling beta-sheet peptide incorporating a hydrolyzable,ester bond (self-assembling depsipeptide; SADP) can be assembled into bilayer beta-sandwich one-dimensional (1D) fibers similarly to conventional beta-sheet peptides. The rate of hydrolysis can be controlled by the pH, temperature, and structural characteristics of the ester unit. The ID fiber of the SADP transforms into vesicle-like 3D structures when the hydrophilic cell-penetrating peptide segment is attached to the SADP segment. Efficient cell internalization of the 3D nanostructures was observed, and we verified the intracellular degradation and disassembly of the biodegradable nanostructures. This study illustrates the potential of biodegradable cross-beta SPNs and provides a valuable toolkit that can be used with self assembling peptides.