Background and Purpose Bladder pain syndrome/interstitial cystitis (BPS/IC) has been clinically treated with glycosaminoglycan (GAG) replenishment therapy. [1, 2] This study was designed to further understand the physiological mechanism behind chondroitin sulfate (CS) treatment and to determine the effect of CS-therapy on recovery of urothelial barrier in an in-vitro chronic injury model. Experimental Approach In differentiated porcine urothelial cells the functional barrier was measured by transepithelial electrical resistance (TEER). A chronic urothelium was inflicted by multiple protamine instillations (3/day for 3 days), to approximate BPS/IC urothelium conditions. CS was instilled afterwards. Recovery of barrier function was followed in time. Additional analyses were performed for immunohistochemistry for barrier markers (tight junctions, GAG’s, umbrella cells) and scanning electron microscopy. Statistics were described by means ± standard error, α = 0.05. Key Results Barrier recovery (TEER) improved significantly with CS instillations compared to protamine only (T=7, 899.1 [Ω.cm2] versus 589.6 [Ω.cm2], p<0.001, 95% CI -394;-255). This recovery effect was seen on all three days and resulted in a significantly higher average TEER value in the CS group after 3 days (2606 Ω.cm2 vs 750.5 Ω.cm2). Immunohistochemistry and scanning electron microscopy showed decreased barrier markers after protamine treatment and enhanced recovery of urothelial GAG’s and other barrier markers after therapeutic instillations. Conclusion and Implications GAG replenishment with CS can improve recovery of barrier function of chronically damaged urothelium in-vitro. This preclinical study supports the hypothesis behind the use of clinical GAG replenishment therapy for patients with a chronically impaired urothelium.