Osteogenic differentiation and bone formation are tightly regulated by several factors, including microRNAs (miRNAs). However, miRNA expression patterns and function during mechanical loading‐induced osteogenic differentiation of human periodontal ligament cells (PDLCs) remain unclear. Here, we investigated the differential expression of miRNA‐195‐5p in the periodontal tissues of mice under orthodontic mechanical loading and in primary human PDLCs exposed to a simulated tension strain. The miR‐195‐5p was observed to be down‐regulated and negatively correlated with osteogenic differentiation. Overexpression of miR‐195‐5p significantly inhibited PDLC differentiation under cyclic tension strain (CTS), whereas the functional inhibition of miR‐195‐5p yielded an opposite effect. Further experiments confirmed that WNT family member 3A (WNT3A), fibroblast growth factor 2 (FGF2), and bone morphogenetic protein receptor‐1A (BMPR1A), proteins important for osteogenic activity and stability, were direct targets of miR‐195‐5p. Mechanical loading increased the WNT3A, FGF2, and BMPR1A protein levels, while miR‐195‐5p inhibited WNT3A, FGF2, and BMPR1A protein expression. WNT, FGF, and BMP signaling were involved in osteogenic differentiation of PDLCs under CTS. Further study confirmed that reintroduction of WNT3A and BMPR1A can rescue the inhibition of miR‐195‐5p on osteogenic differentiation of PDLCs. Our findings are the first to demonstrate that miR‐195‐5p is a mechanosensitive gene that plays an important role in mechanical loading‐induced osteogenic differentiation and bone formation. During orthodontic treatment, periodontal ligament cells can differentiate into osteoblasts under tension strain. The miR‐195‐5p was down‐regulated under tension strain, leading to the disinhibition of WNT3A, FGF2, and BMPR1A, which associated with othodonitc force induced bone formation.