Aims: The effectiveness of cuttlefish bone powder (CBP) in inducing mineralization and cell migration of the osteoblast cell line (MC3T3-E1) was investigated. Materials and methods: The cytotoxicity of 0.5, 1, 5, 25, 50, 100, or 200 µg/mL CBP on an osteoblast cell line was evaluated using the 3-(4,5-dimethylthiazol-2-yl)—2,5-diphenyltetrazolium bromide (MTT) assay. A Boyden chamber assay was used to evaluate the migration of osteoblasts treated with 0.5, 25, or 100 µg/mL CBP. The mineral deposition of treated cells with CBP in the osteogenic medium was determined on days 7, 14, and 28 using alizarin red staining. The area of the calcified nodules was determined on day 28. Analysis of variance and the Mann–Whitney U test were used to compare the difference between the groups. The alkaline phosphatase (ALP) activity of the target cell treated with 0.5 µg/mL CBP was analyzed by using the ALP colorimetric assay kit. Results: All CBP test concentrations were nontoxic to the osteoblast cells. Cell migration after 16 hours in the 0.5, 25, and 100 µg/mL CBP group was 127, 112, and 113%, respectively, compared with the control. The calcified nodule formation on day 28 was greater than on days 7 and 14 and in control groups. ALP expression was found to increase significantly after 28 days of treatment with CBP. Conclusion: The results demonstrate that CBP induces mineralization and cell migration of the osteoblast cell line and the expression of the ALP enzyme. Clinical significance: The cuttlefish bone is a biomaterial that not only induces cell migration but also mineral deposition. The scaffold made from cuttlefish should be suitable for clinical use for bone regeneration.