The bladder presents an attractive target for topical drug delivery. The barrier function of the bladder mucosa (urothelium) presents a penetration challenge for small molecules and nanoparticles. We found that focal mechanical injury of the urothelium greatly enhances the binding and penetration of intravesically-administered cell-penetrating peptide CGKRK (Cys-Gly-Lys-Arg-Lys). Notably, the CGKRK bound to the entire urothelium, and the peptide was able to penetrate into the muscular layer. This phenomenon was not dependent on intravesical bleeding and was not caused by an inflammatory response. CGKRK also efficiently penetrated the urothelium after disruption of the mucosa with ethanol, suggesting that loss of barrier function is a prerequisite for widespread binding and penetration. We further demonstrate that the ability of CGKRK to efficiently bind and penetrate the urothelium can be applied towards mucosal targeting of CGKRK-conjugated nanogels to enable efficient and widespread delivery of a model payload (rhodamine) to the bladder mucosa.