Background: As the main extracellular matrix (ECM), collagen hydrolyzates have great potential in alleviating osteoporosis by promoting osteogenesis and angiogenesis.This work investigated the key peptide sequences in the prepared collagen hydrolyzate (CH) responsible for osteogenic and angiogenic activities and their mol. mechanisms.Addnl., the protective or restorative effects of CH in osteoporotic mice and the regulation of the CH on osteoporosis-related genes were systematically studied.Methods: To characterize the in vitro osteogenic activity of the CH, we assessed the proliferation, differentiation, and mineralization potential of MC3T3-E1 cells using cell counting kit-8 (CCK-8) assay, alk. phosphatase activity kit, and alizarin red staining assay, resp.The impact of CH on EA.hy926 cells was characterized using cell migration assays and Matrigel culture assays.Subsequently, we identified the peptide sequences within the CH and utilized mol. docking anal. to elucidate the mol. mechanisms of action of the key peptides.Furthermore, we conducted animal experiments and used the micro-computed tomog. (micro-CT) and hematoxylin-eosin staining to verify the impact of the CH on osteoporotic mice.Result: Two peptides, Pep-1 (RGPPGPMGPPGLAGPPGE) and Pep-2 (AGPPGPTGPAGPPGFPGAV), strongly bind to EGFR, indicating their potential role in antiosteoporosis activity.The peptides in the CH obtained by mol. docking that have good interactions with EGFR may be potential anti-osteoporosis peptides.The CH tended to alleviate the weight gain of the mice caused by osteoporosis to a certain extent.Compared with the sham group, the model group experienced a substantial increase in alk. phosphatase (ALP) serum levels.The CH alleviated the high level of ALP in the serum of mice caused by osteoporosis.Conclusions: These results suggested that the CH could alter osteoporosis by activating the PI3K/AKT pathway in OVX mice through multiple pathways.Further investigations of the CH, incorporating the identified bioactive peptides as potential nutraceuticals for osteopathic conditions, are warranted.