One of the strategies to improve the therapeutic potential of stem cells is the use of extracellular matrix as scaffold. This improves their retention, viability and differentiation potential. In addition, pretreatment of stem cells with chemicals that induce differentiation to desired lineage has been shown to be an effective approach in cellular therapy. Demethylating agents, 5-azacytidine and zebularine were reported to induce cardiac differentiation in mesenchymal stem cells (MSCs) by many researchers including our group. In this study, we hypothesized that the combination of zebularine pretreatment of bone marrow MSCs and 3D collagen scaffold could improve cardiac differentiation. In our study, we designed two experimental groups; in the first group, MSCs were first treated with 3μM zebularine for 48h and then seeded onto the collagen scaffold while in the second group, MSCs were first seeded and then treated with zebularine. The treated and control groups were analyzed for the expression of MSC specific marker CD44, and for cardiac differentiation markers in terms of percent cell count and measurement of fluorescence intensity per cell after 21 days of treatment. Although, both treatment groups have shown significant expression of cardiac proteins suggesting that MSCs have been successfully differentiated into cardiomyocytes, the results of the second treatment group were more pronounced. Reduced expression of CD44 (p<0.01 and p<0.05) as well as decrease in its fluorescence intensity (p<0.01 and p<0.001) in groups 1 and 2 respectively, suggests loss of stemness. The expression of early cardiac marker, GATA4 showed significant increase in the number of positive cells (p<0.05 and p<0.01) in group 1 and 2 respectively as well as in the fluorescence intensity per cell (p<0.01 in both groups). In case of cTnT and Nkx2.5, the number of positive cells were significantly increased in both treatment groups (p<0.001) whereas their fluorescence intensities were more significantly increased in group 2 (p<0.05 and p<0.01) than in group 1 (p<0.01 and p<0.001) respectively. It is concluded from this study that zebularine treatment in combination with the 3D collagen scaffold plays a significant role in the cardiac differentiation of MSCs in vitro.