Background and Objectives To seek out the role of mircoRNA (miR)-324-5p-modified adipose-derived stem cells (ADSCs) in post-myocardial infarction (MI) myocardial repair. Methods and Results Rat ADSCs were cultivated and then identified by morphologic observation, osteogenesis and adipogenesis induction assays and flow cytometry. Afterwards, ADSCs were modified by miR-324-5p lentiviral vector, with ADSC proliferation and migration measured. Then, rat MI model was established, which was treated by ADSCs or miR-324-5p-modified ADSCs. Subsequently, the function of miR-324-5p-modified ADSCs in myocardial repair of MI rats was assessed through functional assays. Next, the binding relation of miR-324-5p and Toll-interacting protein (TOLLIP) was validated. Eventually, functional rescue assay of TOLLIP was performed to verify the role of TOLLIP in MI. First, rat ADSCs were harvested. Overexpressed miR-324-5p improved ADSC viability. ADSC transplantation moderately enhanced cardiac function of MI rats, reduced enzyme levels and decreased infarct size and apoptosis; while miR-324-5p-modified ADSCs could better promote post-MI repair. Mechanically, miR-324-5p targeted TOLLIP in myocardial tissues. Moreover, TOLLIP overexpression debilitated the promotive role of miR-324-5p-modified ADSCs in post-MI repair in rats. Conclusions miR-324-5p-modified ADSCs evidently strengthened post-MI myocardial repair by targeting TOLLIP in myocardial tissues.