Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit⁺ electrical pacemakers that express the Ano1/TMEM16A Ca²⁺-activated Cl^- channel, whose functions in the GI tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. Kit^CreERT2/+;Ano1^Fl/Fl mice were treated with tamoxifen or vehicle, and small intestines (mucosa-free) examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knock-outs (cKOs) compared to vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca²⁺ transients in ICC-MY network of cKOs displayed reduced duration compared to highly synchronized controls, and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca²⁺ signal imaged field-of-views was: vehicle-controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+ transients' synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow-waves in cKOs displayed reduced duration and increased inter slow-wave interval, and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow-wave and depolarization, previously reported for neonatal constitutive KOs, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca²⁺ transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca²⁺ transients and slow-wave displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causeslack of slow-wave and desynchronized Ca²⁺ transients.