Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca2+ entry in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor beta 1 (TGFβ1) - Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGFβ1 (5 ng/ml for 15 hours) significantly increased Smad3 phosphorylation as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca2+ channel. Consistently, both immunocytochemistry and immunoblot showed that TGFβ1 significantly increased nuclear translocation of Smad3 which was prevented by pre-treatment with thapsigargin. Importantly, the thapsigargin effect was reversed by Lanthanum (La3+) (5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+ channel. Furthermore, knockdown of Orai1, the pore-forming subunit of store-operated Ca2+ channel, significantly augmented TGFβ1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGFβ1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle siRNA delivery system resulted in marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in glomerulus of mice. Taken together, our results indicate that store-operated Ca2+ entry in MCs negatively regulates the TGFβ1-Smad3 signaling pathway.