The peroxisome proliferator-activated receptor-gamma (PPAR-γ) coactivator-1β (PGC-1β) is a well-established regulator of the β-oxidation of fatty acids and the oxidative phosphorylation in mitochondria. However, the underlying mechanism of PGC-1β action remains elusive. This study reveals that PGC-1β is highly induced during myogenic differentiation and knockdown of endogenous PGC-1β by siRNA leads to a decrease in the expression of several mitochondria-related genes. In consistence, the over-expression of PGC-1β stimulates its target genes such as cytochrome c, ATP synthase β and ALAS-1 by its interaction with two transcriptional factors, NRF-1 and ERRα. The deletion or mutation of NRF-1 and/or ERRα binding sites in target gene promoters attenuates their activation by PGC-1β. Moreover, inhibition of NRF-1 or ERRα by siRNA ablated the aforesaid function of PGC-1β and compromised the oxidative phosphorylation and mitochondrial biogenesis. Taken together, these results confirm the direct interaction of NRF-1 and ERRα with PGC-1β, and their participation in mitochondrial biogenesis and respiration. © 2010 Mitochondria Research Society.