Superoxide (O(2)(⋅-)) is the proximal mitochondrial reactive oxygen species underlying pathology and redox signaling. This central role prioritizes development of a mitochondria-targeted reagent selective for controlling O(2)(⋅-). We have conjugated a mitochondria-targeting triphenylphosphonium (TPP) cation to a O(2)(⋅-)-selective pentaaza macrocyclic Mn(II) superoxide dismutase (SOD) mimetic to make MitoSOD, a mitochondria-targeted SOD mimetic. MitoSOD showed rapid and extensive membrane potential-dependent uptake into mitochondria without loss of Mn and retained SOD activity. Pulse radiolysis measurements confirmed that MitoSOD was a very effective catalytic SOD mimetic. MitoSOD also catalyzes the ascorbate-dependent reduction of O(2)(⋅-). The combination of mitochondrial uptake and O(2)(⋅-) scavenging by MitoSOD decreased inactivation of the matrix enzyme aconitase caused by O(2)(⋅-). MitoSOD is an effective mitochondria-targeted macrocyclic SOD mimetic that selectively protects mitochondria from O(2)(⋅-) damage.