Critical limb ischemia is a devastating manifestation of peripheral arterial disease with no effective strategies for improving morbidity and mortality outcomes. We tested the hypothesis that cellular mitochondrial function is a key component of limb pathology and that improving mitochondrial function represents a novel paradigm for therapy. BALB/c mice were treated with a therapeutic mitochondrial-targeting peptide (MTP-131) and subjected to limb ischemia (HLI). Compared to vehicle control, MTP-131 rescued limb muscle capillary density and blood flow (64.7 ± 11% of contralateral vs. 39.9 ± 4%), and improved muscle regeneration. MTP-131 also increased electron transport system flux across all conditions at HLI day-7. In vitro, primary muscle cells exposed to experimental ischemia demonstrated markedly reduced (~ 75%) cellular respiration, which was rescued by MTP-131 during a recovery period. Compared to muscle cells, endothelial cell (HUVEC) respiration was inherently protected from ischemia (~ 30% reduction), but was also enhanced by MTP-131. These findings demonstrate an important link between ischemic tissue bioenergetics and limb blood flow and indicate that the mitochondria may be a pharmaceutical target for therapeutic intervention during critical limb ischemia.