Gastrointestinal (GI) motility disorders such as irritable bowel syndrome (IBS) can occur when coordinated smooth muscle contractile activity is disrupted, and little is known about the molecular identity of the potassium (K+) channels that promote GI tract relaxation. Thus, elucidating the expression and function of the potassium channels could reveal novel therapeutic targets to treat spasmodic gut disorders. Here we utilise quantitative RT-PCR (qPCR), organ bath pharmacology and immunohistochemistry to determine the expression and contribution of K2P channel subtypes in regulating mouse intestinal motility. For isolated organ bath pharmacology, distal ileum and colon segments (2–3cm) were dissected from adult C57BL/6 mice, mounted on an aerator and suspended from a force transducer in an organ bath containing Krebs solution at 32°C. Tension generated from longitudinal muscle contraction was recorded using LabChart® software (AD Instruments). The effects of a variety mechano-gated K2P channel activators were assessed on sub-threshold 10 μM carbachol (CCh) or 60 mM KCl pre-contracted tissue. Data are displayed as mean ± SEM (n = 6, unless stated) and statistical analysis performed by t-test with significance at p < 0.05. Immunohistochemistry and confocal microscopy was performed on perfusion-fixed (1% PFA) whole-mount preparations of the mouse distal ileum and colon. We first performed a qPCR analysis for 14 K2P channel genes on independent ileum and colon sections from 3 animals. KCNK1 (TWIK-1) and KCNK5 (TASK-2) showed the greatest relative expression both in ileum and colon, with KCNK2 (TREK-1) also prominent in ileum but not colon. Given the pause pharmacology for TWIK-1 and TASK-2, and previous research highlighting TREK-1 functionality in colon (1) we pursued the expression pattern and functional role of mechano-gated K2P channels in gut. 100 μM riluzole caused a significant relaxation in CCh pre-contracted muscle tension in both ileum (37.1 ± 3.9 %, n = 14, p <0.0001) and colon (18.9 ± 4.4 %, n = 11, p =0.0023). Relaxation induced by 60 μM BL-1249 and the structurally related fluflenamic acid (100 μM) differed between ileum and colon; 93.1 ± 1.2 % vs 56.1 ± 4.2 % (p <0.001) and 63.3 ± 6.2 % vs 41.1 ± 3.4 % (p = 0.0108) respectively. Immunoreactivity for the TREK-1 channel was widely distributed across both circular smooth muscle and longitudinal smooth muscle in ileum and colon. Immunoreactivity for the TREK-2 channel was located on neurons of both the myenteric and submucosal plexuses in ileum and colon, and was associated with NOS, Chat and calretinin-immunopositive myenteric plexus neurons. TRAAK channel immunoreactivity was evident on NOS, Chat and calretinin-immunopositive neurons located in both myenteric and submucosal plexus neurons in ileum and colon. These data reveal the molecular and functional diversity of the mechano-gated K2P channels within the mouse GI tract and highlight the K2P channels as potential therapeutic targets for treating GI motility disorders.