Background: Conventional therapeutic approaches for ischemic heart disease have limited benefit in preventing postischemic heart failure. Therefore, stem cell therapy emerges as a hopeful tool for treatment of ischemic heart disease following myocardial infarction. Aim: To standardize a protocol ensuring the best differentiation of bone marrow mesenchymal stem cells (BM-MSCs) into cardiomyocytes by using 5-azacytidine with minimum toxicity. Materials and Methods: BM-MSCs were isolated from the bone marrow of albino rats, characterized by flow cytometry and then divided into five groups: Group I: control group, group II: exposed to differentiating medium containing 10 μmol/L of 5-azacytidine for 24 h, group III: exposed to the differentiating medium for 72 h, group IV: the differentiating medium was renewed twice weekly, group V: daily renewal of the differentiating medium was done. The experiment was terminated after 3 weeks from the induction time. Morphological changes of the cultured cells were examined under a phase contrast microscope. The effect of different duration of 5-aza exposure on BM-MSCs viability was evaluated by cell counting Kit 8 assay. Expression of cardiac troponin I and connexin 43 was measured using Quantitative reverse transcription-PCR. Transmission electron microscope was used to observe ultrastructural features of the induced cells. Morphometric study for comparing the length of the formed myofilaments in the four treated groups was done. Results: The obtained results from groups II and III showed initial limited signs of differentiation towards cardiac lineage. Groups IV and V showed optimal signs of differentiation with better results regarding group V, yet, this was at the expense of the viability of the cultured cells. Conclusion:The use of 5-aza showed a valuable differentiating effect on MSCs towards cardiomyocytes. Prolonged use resulted in promotion of the differentiation. Yet, it was at the expense of the viability of these cells.