Noncanonical Wnt signaling in stem cells is essential to numerous developmental events. However, no prior studies have capitalized on the osteoinductive potential of noncanonical Wnt ligands to functionalize biomaterials in enhancing the osteogenesis and associated skeleton formation. Here, we investigated the efficacy of the functionalization of biomaterials with a synthetic Wnt5a mimetic ligand (Foxy5 peptide) to promote the mechanosensing and osteogenesis of human mesenchymal stem cells by activating noncanonical Wnt signaling. Our findings showed that the immobilized Wnt5a mimetic ligand activated noncanonical Wnt signaling via the up-regulation of Disheveled 2 and downstream RhoA-ROCK signaling, leading to enhanced intracellular calcium level, F-actin stability, actomyosin contractility, and cell adhesion structure development. This enhanced mechanotransduction in stem cells promoted the in vitro osteogenic lineage commitment and the in vivo healing of rat calvarial defects. Our work provides valuable guidance for the developmentally inspired design of biomaterials for a wide array of therapeutic applications.,Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).