The complex pathogenesis of cyclophosphamide-induced hemorrhagic cystitis involves arachidonic acid-derived inflammatory mediators, among them leukotrienes. Montelukast, a leukotriene receptor antagonist, is reported to exert an alleviatory effect in the course of cystitis associated with overactive bladder symptoms. The aim of this study was to verify whether the effect of montelukast is also associated with its influence on autonomic activity. The experiment included 20 rats with cyclophosphamide-induced hemorrhagic cystitis (75 mg/kg, four doses every second day), among them, 10 treated with oral montelukast (10 mg/kg for 8 days) and 10 controls. Time and frequency domain analyses of heart rate variability (HRV) were conducted in all the rats as an indirect measure of their autonomic activity. The montelukast-treated animals showed an increase in root mean square of successive differences (rMSSD), as well as an increase in HRV spectrum total power (TP) and power of very low (VLF) spectral component. This suggests that due to its anti-inflammatory and its anti-leukotriene effect, montelukast improves overall autonomic activity, with no preferential influence on either the sympathetic or parasympathetic part. Furthermore, the increase in VLF corresponds to attenuation of inflammatory response. In conclusion, this study showed that aside from its antagonistic effect on leukotriene receptors, montelukast can also modulate autonomic activity.