Aims: Hemorrhagic cystitis (HC) is a major urotoxic complication of cyclophosphamide (CPA) therapy. This study investigated the uroprotective effect of montelukast on CPA-induced HC, compared to the efficacy of 2-mercapto-ethane sulfonate sodium (MESNA). Main methods: Male albino rats were pretreated with MESNA (40 mg/kg/day, IP) or montelukast (10 mg/kg/day, orally) for three days then received a single dose of CPA (200 mg/kg, IP), 1 h after the last dose, and compared to CPA-treated rats receiving drug vehicle. Age-matched rats were used as controls. Bladders of rats were assessed biochemically, macroscopically and microscopically by light and electron microscope 24 h later. Key findings: CPA injection contributed to increased bladder weight, urothelial ulceration, vascular congestion, hemorrhage, increased collagen deposition and mast cell infiltration, compared to control rats. Montelukast preconditioning suppressed mast cell infiltration and inflammatory mediators to greater extent than MESNA. Also, montelukast enhanced autophagosomes formation in detrusor myocytes and up-regulated the autophagy-related proteins (beclin-1 & LC3-II), likely through inhibition of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Montelukast preconditioning offset the up-regulation of transient receptor potential vanilloid 4 (TRPV4) in urothelial tissue of CPA-treated rats, to greater extent than MESNA. Significance: These results demonstrate the uroprotective effect of montelukast on CPA-induced HC, which ap-pears to be more superior to MESNA. These findings suggest that montelukast can emerge as a novel strategy to protect against CPA-induced urotoxicity