The calcium-binding capacity and osteoblast proliferation and differentiation were studied in Alaska pollock surimi hydrolysate (APSH) using a system that mimics the gastrointestinal digestive system. Evaluation of the calcium absorption-promoting ability of APSH revealed that the best calcium-binding ability was achieved after hydrolysis with a combination of pepsin, α-chymotrypsin, and trypsin, and separation into <3kDa (APSH-I), 3-5kDa (APSH-II), 5-10kDa (APSH-III), and <10kDa (APSH-IV) fractions. Scanning electron microscopy with energy-dispersive X-ray spectroscopy analysis confirmed that the hydrolysate and calcium ions formed a complex. Comparison of the calcium absorption capacity using Caco-2 cells showed that calcium absorption was promoted by these hydrolysates. Measurement of the osteoblast activation revealed higher alkaline phosphatase activity, collagen synthesis, and mineralization effect for the low-molecular-weight hydrolysate (LMH) than for the other hydrolysates. In addition, LMH promoted the expression of osteocalcin, osteopontin, and bone morphogenetic protein-2 and -4, which are hormones related to bone formation. Expression of the Runx2 transcription factor, which regulates the expression of these hormones, also increased. These results suggest that Alaska pollock surimi protein hydrolysates prepared using a system that mimics gastrointestinal hydrolysis may result in better osteoblast proliferation and bone health than those prepared using other proteases.