The aim of the present study was to investigate the effects of advanced glycation end products (AGEs) and berberine hydrochloride (BBR) on the osteogenic differentiation ability of human periodontal ligament stem cells (hPDLSCs) in vitro, and their underlying mechanisms. hPDLSCs were subjected to osteogenic induction and were treated with AGEs or AGEs + BBR. Following varying numbers of days in culture, alkaline phosphatase (ALP) activity assays, ALP staining, alizarin red staining, ELISAs, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses were performed to determine the osteogenic differentiation ability of hPDLSCs; RT-qPCR, western blot analysis, and immunofluorescence staining were conducted to investigate the underlying mechanisms. The canonical Wnt/beta-catenin pathway inhibitor XAV-939 and agonist CHIR-99021 were used to determine the contribution of the canonical Wnt/beta-catenin pathway to differentiation. Treatment with AGEs resulted in reduced ALP activity and Collagen I protein levels, decreased ALP staining, fewer mineralized nodules, and downregulated expression of osteogenic-specific genes [Runt-related transcription factor 2 (Runx2), Osterix, ALP, osteopontin (OPN), Collagen I and osteocalcin (OCN)] and proteins (Runx2, OPN, BSP and OCN); however, BBR partially rescued the AGE-induced decrease in the osteogenic potential of hPDLSCs. Furthermore, AGEs activated the canonical Wnt/beta-catenin signaling pathway and promoted the nuclear translocation of beta-catenin; BBR partially attenuated this effect. In addition, XAV-939 partially rescued the AGE-induced reduction in the osteogenic potential of hPDLSCs, whereas CHIR-99021 suppressed the BBR-induced increase in the osteogenic potential of hPDLSCs. The present study indicated that AGEs attenuated the osteogenic differentiation ability of hPDLSCs, in part by activating the canonical Wnt/beta-catenin pathway; however, BBR attenuated these effects by inhibiting the canonical Wnt/beta-catenin pathway. These findings suggest a role for BBR in periodontal regeneration induced by hPDLSCs in patients with diabetes mellitus.