Recently, interest in fish byproduct-derived bioactive components as potential ingredients for treating and preventing osteoporosis is increasing. In this study, we investigated the antiosteoporotic effects of mackerel Scomber japonicus extracts in MC3T3-E1 preosteoblasts in vitro and ovariectomized rats in vivo. Scomber japonicus neutrase-assisted protein hydrolysate (SJNH) treatment enhanced alkaline phosphatase (ALP) activity and osteoprotegerin (OPG) level, and attenuated receptor activator of nuclear factor κB ligand (RANKL) level in MC3T3-E1 cells. In an osteoporotic animal model, ovariectomized (OVX) rats were administered SJNH (200 mg/kg/day) for 8 weeks. SJNH prevented OVX-induced bone mineral density (BMD) loss and restored bone structural compartment loss based on micro-computed tomography (CT). In addition, SJNH administration normalized bone remodeling by regulating bone regeneration and resorption-related proteins such as ALP, osteocalcin (OC), and tartrate-resistant acid phosphatase 5b (TRACP-5b) as well as calcium contents in serum. Taken together, our findings suggest that SJNH can promote antiosteoporotic effects both in vitro and in vivo. Thus, SJNH might be a potent therapeutic agent or functional food ingredient for preventing osteoporosis.