Signalling by cGMP‐dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKI‐dependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5‐trisphosphate receptor I (IP₃RI)‐associated protein (IRAG), which decreases hormone‐induced IP₃‐dependent Ca²⁺ release. We show now that the targeted deletion of exon 12 of IRAG coding for the N‐terminus of the coiled‐coil domain disrupted in vivo the IRAG–IP₃RI interaction and resulted in hypomorphic IRAG^Δ12/Δ12 mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol‐ and phenylephrine‐contracted smooth muscle strips from colon and aorta, respectively, of IRAG^Δ12/Δ12 mice were not relaxed by cGMP, while cAMP‐mediated relaxation was unperturbed. Norepinephrine‐induced increases in [Ca²⁺]_i were not decreased by cGMP in aortic smooth muscle cells from IRAG^Δ12/Δ12 mice. In contrast, cGMP‐induced relaxation of potassium‐induced smooth muscle contraction was not abolished in IRAG^Δ12/Δ12 mice. We conclude that cGMP‐dependent relaxation of hormone receptor‐triggered smooth muscle contraction essentially depends on the interaction of cGKI–IRAG with IP₃RI.