ABSTRACTFollowing myocardial infarction, heart muscle has a limited capacity of self‐healing. Biological platforms providing the natural biochemical and biophysical cues of the native myocardium might be crucial to address current therapeutic shortcomings. The aim of this study was to assess the effect of decellularized human pericardium (DPc), as a bioactive platform, on viability, attachment, proliferation and differentiation of human cardiac progenitors (CPs), and evaluate the possibility of using DPc as a substitute of tissue culture polystyrene (TCPS) substrate for culturing CPs in vitro. The decellularization process removed more than 99% of the cellular components from Pc, yet well preserved its macro‐/micro‐structure and extracellular collagen and glycosaminoglycan content. DPc supported the viability, attachment, metabolism and proliferation of CPs, and enhanced their differentiation into mature cardiomyocytes compared to TCPS. Decellularized pericardium appeared thus to have a high potential for cardiac cell culture and could be applied as a superior alternative to common TCPS. DPc could be then utilized for further tissue engineering applications. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43255.