Cyclophosphamide (CP) treatment is associated with the risk of haemorrhagic cystitis (HC). Moreover, CP-induced HC is complicated by autonomic nervous system (ANS) dysfunction. Pemirolast is thought to be a mast cell stabiliser that inhibits the release of many inflammatory mediators and sensory neuropeptides, and thus, it may be considered a potential chemoprotective HC agent. The aim of the study was to indirectly estimate the effect of pemirolast in experimental HC by measuring ANS activity with the heart rate variability (HRV) method. In CP-treated rats, we found a decreasing trend of overall autonomic activity, together with an imbalance between the main components, and a dominant very low frequency (VLF) power component. Pemirolast treatment did not improve the total HRV power value or the main non-normalized HRV components. Moreover, CP-HC animals treated with pemirolast displayed a different disproportion of normalized spectral components as compared to both control and CP-HC animals without pemirolast treatment, with the balance between normalized low frequency (nLF) and normalized high frequency (nHF) shifted towards nLF. This finding, together with a relatively high VLF tension, indicates that the pemirolast treatment resulted in high sympathetic activity that may contribute to HC exacerbation; thus, this agent seems to be ineffective in CP-induced HC.