Abstract Royal jelly (RJ), secreted by honeybees, contains major fatty acids such as 10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid, which are considered to contribute to bone metabolism. However, these fatty acids are rapidly metabolized in the liver following ingestion, resulting in 2-decenoic acid (2DA) and sebacic acid (SA), respectively. Therefore, elucidating the roles of these metabolites in bone metabolism is of considerable importance. In this study, we focused and investigated the effects of 2DA and SA on osteoblast differentiation using osteoblast-lineage cells. SA treatment significantly upregulated the expression of osteoblast marker genes whereas 2DA had no apparent effect. In addition, SA enhanced alkaline phosphatase activity, a typical marker of osteoblast differentiation and mineralization.