Aging of organisms leads to a decreased ability of tissue regeneration after injury. The regeneration after induced desquamation with biopolymer chitosan of mice bladder urothelium, which is an important blood-urine barrier, has already been studied, but not in old animals. In our study we induced controlled desquamation of urothelial cells of young and old mice with chitosan. Chitosan is a suitable inducer of desquamation, because it is nontoxic and in combination with antibiotics it has already successfully treated bacterial cystitis of young mice. We analyzed urothelial regeneration processes ex vivo and in vivo of both age groups. The aim of our study was to compare the course of the regeneration in young and old mice in order to determine whether the urothelial function and structure of old mice regenerates as in young mice. Ultimately we wanted to determine whether chitosan would be suitable as an auxiliary therapeutic agent for the treatment of bacterial cystitis also in old mice. With microscopic analysis we did not find any significant structural differences between both age groups, as the urothelium of young and old mice was restored during the observed period. In contrast, the urothelial function of old mice was restored faster than of young mice. We noticed a slight time delay in all regeneration processes of old mice in comparison to young mice. Moreover, differences have been found at the subcellular level and in the expression of some mitochondrial and inflammation associated genes. We have also proven that hitosan triggers short-term acute inflammation in both young and old animals, which passes without consequences. Our findings provide a new perspective on the ability of urothelial regeneration in old mice and suggest the possibility of using chitosan as an auxiliary therapeutic agent in the treatment of cystitis also in old mice, which could be the added value for potencial transfer to clinics in the future.