<p>Pericytes in microvasculature of the bladder suburothelium develop spontaneous Ca²⁺ transients that propagate within the pericyte network. Here, the expression of ANO1 Ca²⁺-activated Cl⁻ channels that serve as smooth muscle pacemaker channels was investigated in mural cells of the mouse bladder microvasculature. The involvement of ANO1 in the generation of propagating spontaneous Ca²⁺ transients or vasocontractions was also examined. Fluorescence immunohistochemistry was utilised to visualise ANO1 expression in different microvascular segments. Intercellular Ca²⁺ dynamics in pericytes of capillaries or pre-capillary arterioles (PCAs) were visualised using NG2-GCaMP6 mice, while Ca²⁺ signals in venular pericytes were detected using Cal-520 fluorescence. Spontaneous or electrical field stimulation (EFS)-induced vascular diameter changes were measured using a video-tracking system. ANO1 expression was greatest in capillary pericytes, followed by pericytes in PCAs or post-capillary venules, while arteriolar smooth muscle cells (SMCs) or venular pericytes exhibited lower ANO1 immunoreactivity. In capillaries where pericytes developed nifedipine-resistant propagating spontaneous Ca²⁺ transients, Ani9 (3&#xa0;µM), the specific ANO1 inhibitor, disrupted the intercellular synchrony of Ca²⁺ transients. In venules, Ani9 disrupted the synchrony of spontaneous Ca²⁺ transients amongst venular pericytes and prevented the generation of spontaneous phasic constrictions (SPCs). Ani9 also diminished EFS-evoked, α-adrenergic venular constrictions. In contrast, Ani9 failed to attenuate EFS-induced, predominantly α-adrenergic constrictions in arterioles where SPCs were absent. Thus, ANO1 appears to play a fundamental role in the generation of propagating spontaneous Ca²⁺ transients in capillary, PCA and venular pericytes. Lack of spontaneous Ca²⁺ transients/SPCs in arterioles may be partly attributable to the low ANO1 expression in arteriolar SMCs.</p>