Bone marrow mononuclear cells (BMMNCs) can be used to treat patients with myocardial infarction, since BMMNCs can differentiate in vitro toward cardiomyogenic lineages when treated with transforming growth factor-β1 (TGF-β1). However, the in vitro cardiomyogenic differentiation culture process is costly and laborious, and the patients should wait during the culture period. In this study, we hypothesize that BMMNCs implanted in cardiomyogenically undifferentiated state to myocardial infarction site would differentiate cardiomyogenically in situ when exogenous TGF-β1 is delivered to the cell implantation site. Heparin-conjugated poly(lactic-co-glycolic acid) nanospheres (HCPNs) suspended in fibrin gel were used as a TGF-β1 delivery system. BMMNCs were labeled with a green fluorescent dye (PKH67) and implanted into the infarction border zone of rat myocardium using fibrin gel containing HCPNs and TGF-β1. BMMNC implantation using fibrin gel and HCPNs without TGF-β1 served as a control. Four weeks after implantation, the expression of cardiomyogenic marker proteins by the implanted BMMNCs was dramatically greater in the TGF-β1 delivery group than in the control group. This method can significantly improve the stem cell therapy technology for myocardial regeneration, since it can remove in vitro cell culture step for cardiomyogenic differentiation prior to cell implantation.