Background— Stromal cell-derived factor-1 (SDF-1) is a chemoattractant of stem/progenitor cells, and several studies have shown that SDF-1 may improve ventricular function after infarction. SDF-1 is cleaved by proteases including matrix metalloproteinase-2 (MMP-2) and CD26/dipeptidylpeptidase-4 (DPP-4), which are activated in injured tissues. Methods and Results— We investigated the biodistribution and functional roles of SDF-1 in experimental ischemia/reperfusion injury in rats. Radiolabeled SDF-1 given by intracoronary injection was selectively concentrated in ischemic myocardium. The enhanced uptake of SDF-1 in ischemic myocardium was not mediated by its receptor, CXCR4. Mass spectrometry and Western analyses showed that SDF-1 was cleaved by DPP-4 in plasma and myocardium, whereas a bioengineered MMP-2/DPP-4–resistant form of SDF-1, SSDF-1(S4V), was highly stable. A single dose of SSDF-1(S4V) exhibited greater potency for cardioprotection than wild-type SDF-1. SSDF-1(S4V) improved cardiac function in rats even after a 3-hour ischemic period. Conclusions— These results show that a single dose of protease-resistant SSDF-1(S4V) after myocardial infarction leads to dramatic improvement in angiogenesis and ventricular function even 3 hours after the onset of ischemia, revealing a simple, clinically feasible approach to prevention of heart failure.