Periodontitis is an inflammatory disease that affects tooth-supporting tissues. Chronic inflammation can progress to periodontitis, which results in loss of alveolar bone. Asarylaldehyde is a potential substance for bone metabolism present in natural compounds. Here, we propose the application of asarylaldehyde in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to prevent bone loss. We investigated the effect of asarylaldehyde on hPDLSCs together with bone differentiation media in vitro. The osteogenic differentiation effect was observed after treatment of hPDLSCs with several concentrations of asarylaldehyde. After 21 days, osteogenic cells were identified by mineralization. We also observed that asarylaldehyde increased the mRNA expression of osteoblast-specific markers in hPDLSCs. Interestingly, asarylaldehyde regulated the levels of alkaline phosphatase (ALP) transcriptional activity through the p38/extracellular-signal-regulated kinase (ERK) signaling pathway. Notably, asarylaldehyde induced hPDLSCs to promote osteogenic differentiation. These results suggest that asarylaldehyde plays a key role in the osteogenic differentiation of hPDLSCs. Asarylaldehyde may be a good candidate for the application of natural compounds in future in periodontal regeneration.