The transcriptional coactivator PPAR gamma coactivator 1 alpha (PGC-1alpha) is a key regulator of metabolic processes such as mitochondrial biogenesis and respiration in muscle and gluconeogenesis in liver. Reduced levels of PGC-1alpha in humans have been associated with type II diabetes. PGC-1alpha contains a negative regulatory domain that attenuates its transcriptional activity. This negative regulation is removed by phosphorylation of PGC-1alpha by p38 MAPK, an important kinase downstream of cytokine signaling in muscle and beta-adrenergic signaling in brown fat. We describe here the identification of p160 myb binding protein (p160MBP) as a repressor of PGC-1alpha. The binding and repression of PGC-1alpha by p160MBP is disrupted by p38 MAPK phosphorylation of PGC-1alpha. Adenoviral expression of p160MBP in myoblasts strongly reduces PGC-1alpha's ability to stimulate mitochondrial respiration and the expression of the genes of the electron transport system. This repression does not require removal of PGC-1alpha from chromatin, suggesting that p160MBP is or recruits a direct transcriptional suppressor. Overall, these data indicate that p160MBP is a powerful negative regulator of PGC-1alpha function and provide a molecular mechanism for the activation of PGC-1alpha by p38 MAPK. The discovery of p160MBP as a PGC-1alpha regulator has important implications for the understanding of energy balance and diabetes.