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This study examined changes in the degree of hydrolysis, peptide yield, digestibility, and hydrolyzed amino acid content during the simulated gastrointestinal digestion of Antarctic krill protein. Furthermore, the in vitro proosteogenic activity of different concentrations of the lyophilized residue from the supernatant of the digestion product at the optimal digestion time was evaluated. The results showed that the hydrolysis degree, peptide yield, digestibility, and hydrolyzed amino acid content in the supernatant from Antarctic krill protein digestion increased significantly with digestion time, reaching an equilibrium after 120 min of intestinal digestion, with a hydrolysis degree of 42.67%, a peptide yield of 35.30% and a digestibility of 82.21%. The supernatants at all digestion time points enhanced the osteogenic activity of mouse MC3T3-E1 osteoblasts, the one obtained after 120 min of intestinal digestion being the most effective. Living/dead cell staining, alkaline phosphatase (ALP) activity assay and alizarin red staining confirmed that the supernatant from intestinal digestion for 120 min had good cellular compatibility, promoted osteoblast differentiation and mineralization and consequently regulated its osteogenic activity by enhancing ALP activity and intracellular calcium deposition. The results of this study provide a theoretical basis for the high-value application of Antarctic krill protein.