Mitochondria are known to be involved in cholestatic liver injury, but the damage and biogenesis of mitochondria in response to the early stage of cholestasis is unknown. A rat model of cholestasis was established by bile duct ligation (BDL), with simultaneous creation of the sham group receiving laparotomy without BDL. A significant decrease of liver peroxisome proliferators-activated receptor gamma coactivator-1alpha, mitochondrial transcriptional factor A (Tfam) and glutathione peroxidase (GPx) mRNA and Tfam protein from 6 to 72 h after BDL was found, which was associated with significant decrease of the glutathione, GPx and catalase activity at 72 h. At 72 h after BDL, mitochondrial DNA copy number reached the lowest level, while caspase 9 and 3 activity, but not caspase 8, Bax, Bcl(2), Fas L and Fas-Fas L complex, were upregulated significantly in the liver homogenates of BDL rats. The apoptotic liver cells appeared in large amounts in the rat liver by 72 h after BDL. Our results indicate that transcriptional regulation of the mitochondrial biogenesis is impaired within a few hours after complete bile duct obstruction, resulting in later mitochondrial dysfunction and consequent cholestatic liver injury via the intrinsic apoptosis pathway.