Cardiac tissue engineering focusing on biomaterial scaffolds incorporating cells from different sources has been explored to regenerate or repair damaged area as a lifesaving approach.The aim of this study was to evaluate the cardiomyocyte differentiation potential of human adipose mesenchymal stem cells (hAD-MSCs) as an alternative cell source on silk fibroin (SF) scaffolds for cardiac tissue engineering. The change in surface morphology of SF scaffolds depending on SF concentration (1-6%, w/v) and increase in their porosity upon application of unidirectional freezing were visualized by scanning electron microscopy (SEM). Swelling ratio was found to increase 2.4 fold when SF amount was decreased from 4% to 2%. To avoid excessive swelling, 4% SF scaffold with swelling ratio of 10% (w/w) was chosen for further studies.Biodegradation rate of SF scaffolds depended on enzymatic activity was found to be 75% weight loss of SF scaffolds at the day 14. The phenotype of hAD-MSCs and their multi-linage potential into chondrocytes, osteocytes, and adipocytes were shown by flow cytometry and immunohistochemical staining, respectively.The viability of hAD-MSCs on 3D SF scaffolds was determined as 90%, 118%, and 138% after 1, 7, and 14 days, respectively. The use of 3D SF scaffolds was associated with increased production of cardiomyogenic biomarkers: alpha-actinin, troponin I, connexin 43, and myosin heavy chain. The fabricated 3D SF scaffolds were proved to sustain hAD-MSCs proliferation and cardiomyogenic differentiation therefore, hAD-MSCs on 3D SF scaffolds may useful tool to regenerate or repair damaged area using cardiac tissue engineering techniques.