BACKGROUND AND PURPOSE: Although expression of Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in bladder is reported, their functional role remains unsettled. Here, we immunolocalized the expression of HCN1 and HCN4 subtype in human bladder and investigated their functional significance. EXPERIMENTAL APPROACH: Bladder procured from ten human organ donors were dissected into mucosa (containing urothelium and submucosa) and detrusor for double immunofluorescence of HCN with markers and isometric tension recordings. Mucosa intact and denuded detrusor strips were stretched to a basal tension of 10 mN for eliciting either tetrodotoxin (TTX) resistant spontaneous contractions or TTX sensitive electrical field stimulated (EFS) evoked contractions or carbachol evoked contractions before and after the addition of HCN blocker, ZD7288 or activator, Lamotrigine. KEY RESULTS: Double immunofluorescence revealed prominent immunolocalization of HCN1 and HCN 4 subtype with calcitonin gene related peptide (CGRP), choline acetyl transferase and gap junction proteins in mucosa and detrusor. Removal of mucosa significantly enhanced the basal tension and the spontaneous contractions upon cumulative addition of ZD7288 but not with Lamotrigine (p<0.05). ZD7288[10nM] did not influence the carbachol response, but in presence of Neostigmine [1µM], significantly enhanced the atropine and TTX sensitive EFS contractions of mucosa intact strips. CONCLUSION & IMPLICATIONS: Overall, HCN channels immunolocalized in mucosa, smooth muscle, gap junctions and nerve fibers exert a tonic constraint on detrusor excitability, enable spatio-temporal integration of evoked contraction and constrain the release of neurotransmitters, respectively. In contrast to the pacemaker role in other organs, findings argue for a non-pacemaking role of HCN channels in bladder.