In a previous study, we discovered that the ethanolic extract of sea buckthorn (Hippophae rhamnoides) fruits exhibited anti-osteoporosis effects both in vitro and in vivo. Through bioassay-guided fractionation, we identified the hexane fraction (HRH) as the active fraction, which was further fractionated using preparative HPLC. Among the resulting six fractions, HRHF4 showed significant activity. In the present study, we focused on the bioassay-guided isolation of bioactive compounds from the HRHF4 fraction. We successfully identified the active HRHF43 fraction, which led us to the isolation of potential bioactive compounds (1-6). The chemical structures of these compounds were determined using NMR data, LC-MS analysis, and HR-ESI-MS data as four triterpenes, ursolic acid (1), uvaol (2), oleanolic aldehyde (3), and ursolic aldehyde (4), together with two fatty acids, methyl linoleate (5) and ethyl oleate (6). To evaluate the efficacy of promoting osteoblast differentiation and the expression of mRNA biomarkers related to osteogenesis, we tested the isolated compounds in the mouse mesenchymal stem cell line, C3H10T1/2. Alkaline phosphate staining demonstrated that triterpenes (1-4) displayed osteogenic activity. Particularly noteworthy, ursolic aldehyde (4) exhibited the most potent effect, showing an 11.2-fold higher activity at a concentration of 10 μg/mL compared to the negative control. Moreover, ursolic aldehyde (4) upregulated the gene expression of bone formation-related biomarkers, including Runx2, Osterix, Alp, and Osteopontin. These findings suggest that the fruit extract of H. rhamnoides may have potential as a nutraceutical for promoting bone health, with ursolic aldehyde (4) identified as an active constituent.