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Oral ingestion of collagen hydrolysate leads to significantly high blood levels of various hydroxyproline (Hyp)-containing oligopeptides, such as Pro-Hyp that has been reported to promote osteoblast differentiation. Using ginger protease, we recently developed a novel type of collagen hydrolysate enriched with X-Hyp-Gly-type tripeptides, which were efficiently absorbed into the blood after oral administration of the ginger-degraded collagen hydrolysate. In the present study, we demonstrated that X-Hyp-Gly promoted the differentiation of MC3T3-E1 pre-osteoblasts more effectively than Pro-Hyp, as evidenced by increases in alkaline phosphatase activity and mineralization. In addition, collagen secretion was enhanced by Ala-Hyp-Gly and Leu-Hyp-Gly, particularly at an early stage of differentiation. We detected the uptake of Ala-Hyp-Gly and Leu-Hyp-Gly as well as Pro-Hyp into MC3T3-E1 cells. These results suggest …