Acute pharmacological activation of adenosine monophosphate (AMP)-kinase using 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) has been shown to improve muscle mitochondrial function by increasing mitochondrial biogenesis. We asked whether prolonged AICAR treatment is beneficial in a mouse model of slowly progressing mitochondrial myopathy (Cox10-Mef2c-Cre), and whether the compensatory mechanism is indeed an increase in mitochondrial biogenesis. We treated the animals for 3 months and found that sustained AMP-dependent kinase activation improved cytochrome c oxidase activity, rescued the motor phenotype and delayed the onset of the myopathy. This improvement was observed whether treatment started before or after the onset of the disease. We found that AICAR increased skeletal muscle regeneration thereby decreasing the levels of deleted Cox10-floxed alleles. We conclude that although increase in mitochondrial biogenesis and other pathways may contribute, the main mechanism by which AICAR improves the myopathy phenotype is by promoting muscle regeneration.