Background: Hemorrhagic cystitis (HC) is the most common adverse effect of cyclophosphamide (CP). We assessed the protective role ofMesna and vitamin D against CP induced HC. Methods: This experiment was performed on 64 rats; CON group (control), CP group (sham cyclophosphamide), M group (Mesna), CL group (low dose calcitriol), CH group (high dose calcitriol), VD group (cholecalciferol), CM group (calcitriol & Mesna), and VDM group (cholecalciferol & Mesna). Results: Mesna and vitamin D revealed a significant decrease in visceral pain score, bladder index, malondialdehyde (MDA) and tumor necrosis factor alpha (TNF-α) levels, caspase expression, and a significant increase in total antioxidant capacity (TAC) versus the CP group. Also, the VD, CH, CM & VDM groups revealed a significant decrease in TNF-α level and caspase, microtubule-associated protein light chain 3-II (LC3-II), and sequestosome-1 (SQSTM1/p62) expression versus the M group. The CH group showed a significant increase in serum calcium versus other groups. Conclusion: Vitamin D increased the uroprotective effect of Mesna, which is attributed to its ability to restore autophagy flux.