Short dwell-time and poor penetration of the bladder permeability barrier (BPB) are the main obstacles to intravesical treatments for bladder diseases, and is evidenced by the lack of such therapeutic options on the market. Herein, we demonstrate that by finely tuning the molecular weight of our cationic polymer mucoadhesive nanoparticles, we enhanced our gene transfer, leading to improved adherence and penetrance through the BPB in a safe and efficient manner. Specifically, increasing the polymer molecular weight from 45 kDa to 83 kDa enhanced luciferase plasmid transfer to the healthy murine bladder, leading to 1.35 ng/g luciferase protein expression in the urothelium and lamina propria regions. The relatively higher molecular weight polymer (83 kDa) did not induce morphologic changes or inflammatory responses in the bladder. This approach of altering polymer molecular weight for prolonging gene transfer residence time and deeper penetration through the BPB could be the basis for the design of future gene therapies for bladder diseases.