Objective: Cardiomyocytes, known as cells incapable of self-renewal when injured, and therefore heartrelated problems, have recently attracted a great deal of attention in regenerative medicine. But what has become more important in cell therapy, is seeking for approaches resulting in more functional cells and therefore, more effective treatments. One of these methods, whose idea has been inspired from the physical environment that the tissues deal with in the body, is mechanotransduction. This study has focused on the comparison of GATA4 (cardiac-specific transcription factor gene) expression between the rabbit mesenchymal stem cells subjected to equiaxial cyclic stretch and the ones not treated mechanically. Materials and Methods: After aspiration of bone marrow from the iliac crest of male rabbits, percoll separation technique was used to extract mesenchymal stem cells. The isolated cells were characterized using flowcytometry method. Their multi-potency was explored by directing them towards adipogenic, osteogenic and chondrogenic differentiation. For cardiomyogenic differentiation, the stem cells seeded on collagen-coated membranes were studied in four groups including undifferentiated (negative control), chemically, mechanically and chemically-mechanically treated cells. Rabbit cardiomyocytes were taken as positive control. After four days, GATA4 expression in each group was quantified using Taqman based Real Time-PCR method. HPRT was taken as housekeeping gene. Results: The results obtained from flowcytometry and multipotency tests validated the extraction process and the stem cells' ability to differentiate, respectively. The Real Time PCR data showed that expression of GATA4 in cells subjected to mechanical-chemical and mechanical treatment was more than its expression in the other two groups. Conclusion: Our results suggest that mechanical loading as a single differentiation factor, affects the differentiation of mesenchymal stem cells into cardiomyocytes. Combination of Chemical factors with mechanical loading may increase the differentiation of stem cells into cardiomyocytes and produce functional cardiomyocytes in engineered tissues.