Intravesical treatment of superficial bladder cancer with epirubicin is associated with local urological adverse effects, the causes of which are unknown. Our aim was to investigate the effects of epirubicin on the release of urothelial mediators and inflammatory cytokines. UROtsa cells were treated with epirubicin for 1 h at 37 °C. Release of urothelial transmitters and inflammatory cytokines was examined immediately, 24 h and 7 days following treatment. Release of ATP and nitric oxide were increased transiently after treatment with epirubicin (0.01 mg/ml), but this was not evident one-week after treatment. Basal prostaglandin E2 release was decreased at 24 h but not at 7 days. An increase in basal acetylcholine release and decrease in stretch-induced acetylcholine release were observed 24-h after treatment (0.01 mg/ml). One week following epirubicin treatment (0.001 mg/ml), stretch-induced, but not basal acetylcholine release, was observed. Secretion of interleukin-6 and interleukin-8 was increased 70-fold and 5-fold respectively, at 24 h (0.01 mg/ml). This was sustained and one week after epirubicin treatment (0.001 mg/ml), the increase in the secretion of both inflammatory cytokines was still evident. Epirubicin treatment induces several transient changes in urothelial function. However, the increased secretion of inflammatory cytokines (IL-6 and IL-8) is sustained and these mediators may be involved in the pathophysiology of bladder toxicity following intravesical epirubicin treatment.