The effects of substrate stiffness on the development of cardiomyocytes have been investigated extensively. Polydimethylsiloxane (PDMS) elastomer is one of biomaterials that are commonly used to explore the effects of substrate compliance on stem cell differentiation. Although the effects of substrate stiffness on cardiac differentiation of pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have been reported, whether the stiffness of PDMS-based substrates could enhance differentiation of iPSCs toward cardiomyocyte lineage or not remains unknown. In this study, we found that a denser gelatin distribution and a higher gelatin adsorption on the stiffer PDMS. In addition, nanotopographies on PDMS substrates with different stiffness were distinct. iPSCs on the stiffer PDMS substrates showed higher pluripotency marker but lower cardiac gene expressions. In contrast, iPSCs on the softer PDMS substrates revealed lower pluripotency marker but higher cardiac gene expressions. These results indicate that stiffer PDMS substrates with gelatin coating could be used to support iPSC self-renewal and softer PDMS substrates coated with gelatin could be used for enhanced cardiac differentiation of iPSCs.