Key points The mucosa may have neuron‐like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established. The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated. The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+. These spontaneous events primarily depend on Ca2+ handling by sarco‐endoplasmic reticulum Ca2+ stores. A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco‐endoplasmic reticulum Ca2+ handling. The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances. Abstract The role of the mucosa in generating the spontaneous activity of guinea‐pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi‐fluorescence Ca2+ imaging, respectively. Mucosa‐intact but not mucosa‐denuded SV preparations generated TTX‐ (1 μm) resistant spontaneous phasic contractions that were abolished by nifedipine (3 μm). Consistently, SMCs developed mucosa‐dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10 μm) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10 μm), a sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor. DIDS (300 μm) and niflumic acid (100 μm), blockers for Ca2+‐activated Cl− channels (CACCs), or low Cl− solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L‐type voltage‐dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco‐endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances.