• Lack of TRIC-B in osteoblasts strongly impairs skeleton growth and structure, leading to bone fractures. • In absence of TRIC-B, the impaired Ca2+ flux from endoplasmic reticulum to cytosol is associated to delayed osteoblast differentiation and decreased collagen synthesis that cause reduced collagen incorporation in the extracellular matrix and poor mineralization. • An impaired SMAD signaling in mutant mice and in OI patient osteoblasts explains the osteoblast malfunction. • The reduced SMAD phosphorylation and nuclear translocation are mainly caused by alteration in Ca2+ calmodulin kinase II (CaMKII)-mediated signaling. Ca2+ is a second messenger that regulates a variety of cellular responses in bone, including osteoblast differentiation. Mutations in trimeric intracellular cation channel B (TRIC-B), an endoplasmic reticulum channel specific for K +, a counter ion for Ca2+flux, affect bone and cause a recessive form of osteogenesis imperfecta (OI) with a still puzzling mechanism. Using a conditional Tmem38b knock out mouse, we demonstrated that lack of TRIC-B in osteoblasts strongly impairs skeleton growth and structure, leading to bone fractures. At the cellular level, delayed osteoblast differentiation and decreased collagen synthesis were found consequent to the Ca2+ imbalance and associated with reduced collagen incorporation in the extracellular matrix and poor mineralization. The impaired SMAD signaling detected in mutant mice, and validated in OI patient osteoblasts, explained the osteoblast malfunction. The reduced SMAD phosphorylation and nuclear translocation were mainly caused by alteration in Ca2+ calmodulin kinase II (CaMKII)-mediated signaling and to a less extend by a lower TGF-β reservoir. SMAD signaling, osteoblast differentiation and matrix mineralization were only partially rescued by TGF-β treatment, strengthening the impact of CaMKII-SMAD axes on osteoblast function. Our data established the TRIC-B role in osteoblasts and deepened the contribution of the CaMKII-SMAD signaling in bone. [ABSTRACT FROM AUTHOR]