The role of the extracellular matrix (ECM) in guiding cell fate has spurred the development of synthetic, ECM-inspired regenerative biomaterials. We previously described a porous, open-cell foam composed of nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG), capable of inducing in vitro osteogenesis of primary human mesenchymal stem cells (hMSCs) and in vivo skull regeneration without adding growth factors or expanded progenitor cells. This study investigated the role of store-operated calcium entry (SOCE) in MC-GAG activity by evaluating the impact of Orai1, the pore-forming subunit of the calcium release-activated channel. Compared to nonmineralized collagen glycosaminoglycan (Col-GAG), hMSCs cultured on MC-GAG exhibited increased Orai1 expression. Chemical inhibition of SOCE using MRS1845 reduced the expression of both gene and protein of osteogenic markers, as well as mineralization on MC-GAG, while MRS1845 exerted no effects on Col-GAG materials. Orai1 knockdown similarly inhibited matrix mineralization and expression of the late osteogenic marker, bone sialoprotein-2 (BSP2), on MC-GAG, whereas no effects were notable on Col-GAG. Orai1 knockdown reduced ERK1/2 phosphorylation, suggesting that the osteogenic effects of Orai1 functioned downstream of ERK1/2 pathways. These findings suggest Orai1 is necessary for osteoprogenitor maturation and matrix mineralization in MC-GAG-mediated osteogenic differentiation.