Matrix protein is a kind of secretory protein that regulates the biomineralization of the bivalve shell. In this study, a water-soluble matrix protein (WSMP) from Pacific oysters (Crassostrea gigs) shell was isolated, and its structure was analyzed in detail, in addition to its anti-osteoporosis activity in vitro and in vivo. Results showed that WSMP was an acidic protein with an apparent molecular mass of 47 and 79 kDa and contained a glycoprotein structure. In vitro, the reduction of Tartrate-resistant acid phosphatase (TRAP) and deoxypyridinoline (DPD) indicated that osteoclast activity was inhibited compared with the model group. Moreover, the increased osteocalcin (OCN) and BMD levels suggested that the high osteoblast activity and bone mineralization was improved. SEM analysis of the femur showed that there were fewer bone pits in experimental groups, which was consistent with the above results. In vivo, WSMP promoted the expression of alkaline phosphatase (ALP) and osteogenic differentiation factor BMP-2 in osteoblasts. In addition, the activity of osteoclasts was inhibited by regulating the process of osteoclast differentiation induced by RANKL. Both in vitro and in vivo studies showed that WSMP could promote osteogenesis and inhibit osteoclast absorption, thus demonstrating their potential applications in osteoporosis.