Objective@#To investigate the effect of microRNA-26a-5p on osteogenic differentiation of human periodontal ligament stem cells (hPDLSC) and its related mechanisms.@*Methods@#hPDLSC in periodontal tissues from healthy adults and hPDLSC from periodontitis patients (PPDLSC) were isolated and cultured in vitro, respectively. The PPDLSC were divided into Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ groups. Group Ⅰ is control group, and the other four groups were transiently transfected with miR-NC, miR-26a-5p, antimiR-NC and antimiR-26a-5p lentiviral vectors, respectively. The osteogenic differentiation abilities of the cells in vitro were determined by alizarin red staining, alkaline phosphatase (ALP) activity assay and real-time quantitative PCR (qPCR). Totally 40 male mice (6-weeks) were equally divided into five groups with 8 mice in each group. The PPDLSCs cells (1×107/ml) in Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ groups, which adhered to hydroxyapatine-tricalcium phosphate (HA-TCP), were implanted into the nude mice subcutaneously and the animal models were constructed to analyze the effect of miR-26a-5p on the osteogenic differentiation of PPDLSCs in vivo. PPDLSCs were divided into A, B, C, D groups, and transfected with miR-26a-5p+Wnt5a-Wt, miR-NC+Wnt5a-Wt, miR-26a-5p+Wnt5a-Mut and miR-NC+Wnt5a-Mut in each of the above mentioned 5 groups, respectively. The luciferase activity assay was used to detect the relative luciferase in A, B, C and D groups to analyze the targeting relationship between miR-26a-5p and Wnt5a. Osteogenic differentiation related proteins expression were analyzed by western blotting.@*Results@#hPDLSC and PPDLSC were observed consistent with the characteristics of mesenchymal stem cells and had osteogenic differentiation ability in vitro. Compared with hPDLSC [(89.87±8.12)%], the osteogenic capacity of PPDLSC [(31.46±6.56)%] was significantly lower (P<0.05). The ALP activity (1.88±0.59), calcified nodules (79.88±5.92), the expression of the osteogenic differentiation markers Runt-related transcription factor 2 (Runx2) (2.40±0.70), ALP (2.10±0.60) and osteocalcin (3.00±0.90) mRNA in the PPDLSC from Group Ⅲ were significantly higher in comparison with the control group [(0.88±0.34), (29.69±2.65), (1.30±0.30), (0.09±0.25), (1.71±0.50)], while those from Group Ⅴ[(0.44±0.07), (14.83±3.05), (0.50±0.11), (0.30±0.08) and (0.80±0.17)] were significantly lower (P<0.05). In vivo studies in nude mice showed that the proportion of the osteogenic region [(34.96±5.65)%] in the miR-26a-5p group was significantly increased in comparison with the control group [(23.28±3.03)%], while in the antimiR-26a-5p group [(8.02±2.27)%] was significantly lower (P<0.05). The luciferase activity of the Group A (0.46±0.06) was significantly lower than Group B (3.46±0.45) (P<0.05). Compared with the control group, the expression levels of Wnt5a protein, calmodulin kinase Ⅱ and protein kinase C proteins in the Group Ⅲ were significantly decreased, while those in the GroupⅤ were significantly increased (P<0.05).@*Conclusions@#MicroRNA-26a-5p could promote osteogenic differentiation of PPDLSC in vivo and in vitro, and its mechanism might be inhibiting the activation of Wnt/Ca2+ signaling pathway by targeting Wnt5a.