Hemorrhagic cystitis is one of the most important complications of cyclophosphamide, a drug widely used in cancer chemotherapy and bone marrow transplantation. 5-HT antagonists are anti-emetic agents and have been shown to have notable anti-inflammatory and antioxidant properties. This study was designed to investigate the possible protective effects of tropisetron against cyclophosphamide-induced hemorrhagic cystitis in rats. Hemorrhagic cystitis was induced in female rats by cyclophosphamide (270 mg/kg). Tropisetron (2.5, 5 and 7.5 mg/kg), granisetron (2.5 and 5 mg/kg), and ondansetron (5 mg/kg) were injected 15 min before, 4 and 8 h after cyclophosphamide. To evaluate the role of alpha7 nicotinic acetylcholine receptor (α7nAChR), its antagonist, methyllycaconitine (5 mg/kg) was administered 30 min before tropisetron. After 24 h, animals were killed under anesthesia. Macroscopic and histological changes were evaluated. Malondialdehyde (MDA), glutathione (GSH) and Evans blue were measured spectrophotometrically. Furthermore, the protein levels of p38 mitogen-activated protein kinases (P38 MAPK), p-P38, signal transducer and activator of transcription 3 (STAT3), p-STAT3 and Poly (ADP-ribose) polymerase (PARP) were determined using Western blot. Cyclophosphamide administration significantly induced histopathological damages and increased MDA, p-p38/p38, p-STAT3/STAT3, and PARP levels compared with the saline group. Tropisetron treatment diminished histopathological injuries as well as MDA level, and STAT3 activity compared to cyclophosphamide treated rats. Co-administration of methyllycaconitine with tropisetron, partially or completely reversed the protective effects of tropisetron. Our results showed that prophylactic administration of tropisetron markedly ameliorated the cyclophosphamide-induced bladder hemorrhage and inflammation in rats. These effects of tropisetron were α7nAChR dependent.