Substrate stiffness has been indicated as a primary determinant for stem cell fate, being capable of influencing motility, proliferation, and differentiation. Although the effects of stiffness on cardiac differentiation of human-induced pluripotent stem cells (h-iPSCs) have been reported, whether stiffness of polydimethylsiloxane-based substrates could enhance differentiation of h-iPSCs toward heart valve endothelial cells lineage (VECs) or not remains unknown. Herein, we modulated the substrate stiffness to evaluate its effect on the differentiation of h-iPSCs into valve endothelial-like cells (h-iVECs) in vitro and determine the suitable stiffness. The results revealed that VECs-related genes (PECAM1, CDH5, NFATC1, etc.) were significantly increased in h-iVECs obtained from the three substrates compared with h-iPSCs. Gene expression levels and differentiation efficiency were higher in the medium group than in the stiff and soft groups. An increase in substrate stiffness to 2.8 GPa decreased the efficiency of h-iPSCs differentiation into h-iVECs and downregulated VECs specific genes. Through mRNA sequencing, we determined the key genetic markers involved in stiffness guiding the differentiation of cardiac progenitor cells into h-iVECs. Unsupervised hierarchical clustering showed that medium stiffness were more suitable for the differentiation of h-iPSCs into h-iVECs in vitro. Moreover, this process is regulated by the WNT/Calcineurin signaling pathway. Overall, this study demonstrates how stiffness can be used to enhance the h-iVECs differentiation of iPSCs and emphasizes the importance of using substrate stiffness to accomplish a more specific and mature differentiation of h-iVECs for future therapeutic and tissue engineering valve applications. Statement of significanceSeveral studies have examined the stiffness-induced cell fate from pluripotent stem cells during the stage of mesoderm cell differentiation. This is the first research that rigorously examines the effect of substrate stiffness on human valve endothelial-like cells differentiation from cardiac progenitor cells. We found that the medium stiffness can increase the differentiation efficiency of h-iVECs from 40% to about 60%, and this process was regulated by the WNT/CaN signaling pathway through the activation of WNT5a. Substrate stiffness not only increases the differentiation efficiency of h-iVECs, but also improves its cellular functions such as low-density lipoprotein uptake and NO release. This study emphasizes the importance of using substrate stiffness to accomplish a more specific and mature differentiation of h-iVECs.