Inositol 1,4,5triphosphate receptorassociated cGMP kinase substrate 1 (IRAG1) is a substrate protein of the NO/cGMP-signaling pathway and forms a ternary complex with the cGMPdependent protein kinase Iβ (PKGIβ) and the inositol triphosphate receptor I (IP3RI). Functional studies about IRAG1 exhibited that IRAG1 is specifically phosphorylated by the PKGIβ, regulating cGMPmediated IP3dependent Ca2+release. IRAG1 is widely distributed in murine tissues, e.g., in large amounts in smooth muscle-containing tissues and platelets, but also in lower amounts, e.g., in the spleen. The NO/cGMP/PKGI signaling pathway is important in several organ systems. A loss of PKGI causes gastrointestinal disorders, anemia and splenomegaly. Due to the similar tissue distribution of the PKGIβ to IRAG1, we investigated the pathophysiological functions of IRAG1 in this context. Global IRAG1KO mice developed gastrointestinal bleeding, anemiaassociated splenomegaly and iron deficiency. Additionally, Irag1deficiency altered the protein levels of some cGMP/PKGI signaling proteins—particularly a strong decrease in the PKGIβ—in the colon, spleen and stomach but did not change mRNAexpression of the corresponding genes. The present work showed that a loss of IRAG1 and the PKGIβ/IRAG1 signaling has a crucial function in the development of gastrointestinal disorders and anemia-associated splenomegaly. Furthermore, global Irag1deficient mice are possible in vivo model to investigate PKGIβ protein functions.