Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of cerebral ischemia. In this study, we explored whether MMP activity can be visualized by noninvasive near-infrared fluorescence (NIRF) imaging using an MMP-activatable probe in a mouse model of stroke. C57Bl6 mice were subjected to transient middle cerebral artery occlusion (MCAO) or sham operation. Noninvasive NIRF imaging was performed 24 h after probe injection, and target-to-background ratios (TBRs) between the two hemispheres were determined. TBRs were significantly higher in MCAO mice injected with the MMP-activatable probe than in sham-operated mice and in MCAO mice that were injected with the nonactivatable probe as controls. Treatment with an MMP inhibitor resulted in significantly lower TBRs and lesion volumes compared to injection of vehicle. To test the contribution of MMP-9 to the fluorescence signal, MMP9-deficient (MMP9(-/-)) mice and wild-type controls were subjected to MCAO of different durations to attain comparable lesion volumes. TBRs were significantly lower in MMP9(-/-) mice, suggesting a substantial contribution of MMP-9 activity to the signal. Our study shows that MMP activity after cerebral ischemia can be imaged noninvasively with NIRF using an MMP-activatable probe, which might be a useful tool to study MMP activity in the pathophysiology of the disease.