Objective— To investigate the effects of pioglitazone (PIO), a peroxisome proliferator–activated receptor γ agonist, on plaque matrix metalloproteinase (MMP) and macrophage (Mac) responses in vivo in a molecular imaging study. Methods and Results— In vitro, PIO suppressed MMP-9 protein expression in murine peritoneal Macs ( P <0.05). To assess PIO’s effects on plaque inflammation, nondiabetic apolipoprotein E ^−/− mice receiving a high-cholesterol diet (HCD) were administered an MMP-activatable fluorescence imaging agent and a spectrally distinct Mac-avid fluorescent nanoparticle. After 24 hours, mice underwent survival dual-target intravital fluorescence microscopy of carotid arterial plaques. These mice were then randomized to HCD or HCD plus 0.012% PIO for 8 weeks, followed by a second intravital fluorescence microscopy study of the same carotid plaque. In the HCD group, in vivo MMP and Mac target-to-background ratios increased similarly ( P <0.01 versus baseline). In contrast, PIO reduced MMP and Mac target-to-background ratios ( P <0.01) versus HCD. Changes in MMP and Mac signals correlated strongly ( r ≥0.75). Microscopy demonstrated MMP and Mac reductions in PIO-treated mice and a PIO-modulated increase in plaque collagen. Conclusion— Serial optical molecular imaging demonstrates that plaque MMP and Mac activity in vivo intensify with hypercholesterolemia and are reduced by PIO therapy.