p53 is a tumor suppressor protein that also plays a role in regulating aerobic metabolism. Since skeletal muscle is a major source of whole body aerobic respiration, it is important to delineate the effects of p53 on muscle metabolism. In p53 knockout (KO) mice, we observed diminished mitochondrial content in mixed muscle and lowered PGC-1 protein levels in gastrocnemius muscle. In the intermyofibrillar (IMF) mitochondria, lack of p53 was associated with reduced respiration and elevated ROS production. Permeability transition pore (mtPTP) kinetics remained unchanged, however IMF mitochondrial cytochrome c release was reduced and DNA fragmentation was lowered, illustrating a resistance to mitochondrially-driven apoptosis in muscle of KO mice. p53 null animals displayed similar muscle strength, but greater fatigability and less locomotory endurance than wild-type (WT) animals. Surprisingly, the adaptive responses in mitochondrial content to running were similar in both WT and KO mice. Thus p53 may be important, but not necessary for exercise-induced mitochondrial biogenesis. In WT animals, acute muscle contractions induced the phosphorylation of p53 in concert with increased activation of upstream kinases AMPK and p38, indicating a pathway through which p53 may initiate mitochondrial biogenesis in response to contractile activity. These data illustrate a novel role for p53 in maintaining mitochondrial biogenesis, apoptosis and performance in skeletal muscle.