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ABSTRACT ObjectiveThis study aimed to investigate the potential role of cesium chloride (CsCl), ivabradine (IVA), and isoproterenol (ISO) on the sensory transmission of bladder afferents to graded urinary bladder distension (UBD). We specifically selected these drugs to target the hyperpolarization‐activated cyclic nucleotide‐gated (HCN) cation channels to determine their role in afferent encoding. MethodsThe bladders of C57BL/6 female mice were harvested with attached pelvic nerves in continuity, and the stimulus–response function (SRF) of bladder afferents to stepped bladder distension (20, 40, 60, 80 cmH₂O) was recorded by single‐fiber recordings. Their changes in SRF to bath application of CsCl, IVA, and ISO were then evaluated. The presence of HCN on bladder afferent endings was assessed through immunohistological staining on bladder sections from mice with genetically labeled bladder afferents. ResultsIVA and ISO did not significantly reduce afferent responses to UBD, whereas CsCl increased afferent responses. Bladder afferents in the pelvic nerve pathway were categorized into low‐firing (LF, < 10 Hz) and high‐firing (HF, > 10 Hz) groups. SRF in both the LF and HF groups showed similar trends with no significant changes in response to IVA and ISO. CsCl increased SRF only in the HF group but not in the LF group. Immunohistological staining revealed that HCN1 does not extensively co‐localize with afferent endings, showing only sporadic presence. ConclusionOur targeted pharmacological studies with single‐fiber recordings and immunohistological staining collectively suggest that HCN channels do not play a significant role in bladder afferent sensory transmission.