Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca(2+) channels (VDCCs) or TMEM16A Ca(2+)-activated Cl(-) channels significantly changes global cytosolic Ca(2+) concentration ([Ca(2+)]i) and the tone. TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca(2+)]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca(2+)]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence.