Endoplasmic reticulum (ER) stress is an emerging factor in fibrotic disease, although precise mechanisms are not clear. Calreticulin (CRT) is an ER chaperone and regulator of Ca2+ signaling up-regulated by ER stress and in fibrotic tissues. Previously, we showed that ER CRT regulates type I collagen transcript, trafficking, secretion, and processing into the extracellular matrix (ECM). To determine the role of CRT in ECM regulation under fibrotic conditions, we asked whether CRT modified cellular responses to the pro-fibrotic cytokine, TGF-β. These studies show that CRT-/- mouse embryonic fibroblasts (MEFs) and rat and human idiopathic pulmonary fibrosis lung fibroblasts with siRNA CRT knockdown had impaired TGF-β stimulation of type I collagen and fibronectin. In contrast, fibroblasts with increased CRT expression had enhanced responses to TGF-β. The lack of CRT does not impact canonical TGF-β signaling as TGF-β was able to stimulate Smad reporter activity inCRT-/- MEFs. CRT regulation of TGF-β-stimulated Ca 2+ signaling is important for induction of ECM. CRT-/- MEFs failed to increase intracellular Ca2+ levels in response to TGF-β. NFAT activity is required for ECM stimulation by TGF-β. In CRT-/- MEFs, TGF-β stimulation of NFAT nuclear translocation and reporter activity is impaired. Importantly, CRT is required for TGF-β stimulation of ECM under conditions of ER stress, as tunicamycin-induced ER stress was insufficient to induce ECM production in TGF-β stimulated CRT-/- MEFs. Together, these data identify CRT-regulated Ca2+-dependent pathways as a critical molecular link between ER stress and TGF-β fibrotic signaling. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.