A Thunnus orientalis bone-based collagen hydrolysate (TBCH) was prepared with alcalase, and its osteogenic effect was examined using MC3T3-E1 cells. The mechanism by which TBCH promotes osteogenesis was also investigated. The results showed that TBCH, even at an extremely low concentration (2 mu g/mL), significantly stimulated the proliferation and promoted osteogenic differentiation of MC3T3-E1 cells. This suggests that TBCH is biocompatible and potentially useful for promoting bone formation. Moreover, TBCH significantly upregulated the mRNA levels of osteogenic markers, such as runt-related transcription factor 2, alkaline phosphatase, osteopontin, and osteocalcin. Western blot results showed that the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway in MC3T3-E1 cells was activated by treatment with TBCH. Based on these results, we speculate that TBCH could provide a promising way for inducing bone regeneration. (C) 2019 Elsevier Ltd. All rights reserved.