The urothelium is a vital permeability barrier that prevents the uncontrolled flow of urinary components into and out of the bladder interstitium. Our study addressed the question of possible sex-specific variations in the urothelium of healthy mice and their impact on chronic bladder inflammation. We found that healthy female bladders have a less robust barrier function than male bladders, as indicated by significant differences in transepithelial electrical resistance (TEER) values. These differences could be attributed to detected higher claudin 2 mRNA expression and a less pronounced glycocalyx in females than in males. In addition, TEER measurements showed delayed barrier recovery in chronically inflamed female bladders. We found subtle differences in the expressions of genes involved in the regulation of the actin cytoskeleton between the sexes, as well as pronounced urothelial hyperplasia in females compensating for attenuated barrier function. The identified genetic variations in glycosylation pathways may also contribute to this divergence. Our findings add to the growing body of literature on the intricate sex-specific nuances of urothelial permeability function and their implications for chronic bladder inflammation. Understanding these differences could lead to tailored diagnostic and therapeutic approaches in the treatment of bladder disorders in the future.