Micro/nano-topographies (MNTs) of biomaterials are well-known to influence osteoblast differentiation, although the underlying mechanisms remain incompletely understood. Piezo1 is involved in mechanotransduction in various cell types, and we hypothesized that Piezo1 may play a key role in osteogenic differentiation through the NFATc1-Wnt/β-catenin or YAP signaling pathways. To test this hypothesis, polished titanium plates (PT) and titanium plates with TiO2 nanotubes (TNT-30 and TNT-100) were used. Experimental results showed that TNT-100 increased the gene expression and activation of Piezo1, upregulating osteogenic genes such as NFATc1, Wnt3a, and β-catenin, and promoting the nuclear localization of NFATc1, β-catenin, and YAP compared to PT. The influx of Ca2+ through Piezo1, activated by MNTs, initiated the signaling cascades. Furthermore, compared to TRPV4, another recently reported mediator in MNTs-stimulated osteogenic differentiation, Piezo1 responded to MNTs much faster. These findings suggest that Piezo1 acts as an early mediator of osteogenic differentiation stimulated by MNTs in BMSCs.© 2025 Wiley Periodicals LLC.