Disruption of the blood-urine barrier can result in acute or chronic inflammatory bladder injury. Activation of the oxygen-regulated hypoxia-inducible factor (HIF) pathway has been shown to protect mucosal membranes by increasing the expression of cytoprotective genes and by suppressing inflammation. The activity of HIF is controlled by prolyl hydroxylase domain (PHD) dioxygenases, which have been exploited as therapeutic targets for the treatment of anemia of chronic kidney disease. Here we established a mouse model of acute cyclophosphamide (CYP)-induced blood-urine barrier disruption associated with inflammation and severe urinary dysfunction to investigate the HIF-PHD axis in inflammatory bladder injury. We found that systemic administration of dimethyloxalylglycine (DMOG) or molidustat, two small molecule inhibitors of HIF-prolyl hydroxylases (HIF-PHIs), profoundly mitigated CYP-induced bladder injury and inflammation as assessed by morphologic analysis of transmural edema and urothelial integrity and by measuring tissue cytokine expression. Void spot analysis to examine bladder function quantitatively demonstrated that HIF-PHI administration normalized micturition patterns and protected against CYP-induced alteration of urinary frequency and micturition patterns. Our studies highlight the therapeutic potential of HIF-activating small molecule compounds for the prevention or therapy of bladder injury and urinary dysfunction due to blood-urine barrier disruption.