Residual feed intake (RFI) is a popular measure of feed efficiency in livestock animals. The functionality of mitochondrial metabolism has been shown to be associated with variation in feed efficiency. In this study, mitochondrial biogenesis was determined by measuring the mitochondrial DNA (mtDNA) copy number in skeletal muscle of two groups of fat-tailed lambs divergently ranked on phenotypic expression of RFI (high- and low-RFI). A quantitative real-time PCR was performed to determine the relative mRNA levels of peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) and NAD+-depended deacetylase sirtuin1 (SIRT1), as two major genes involved in the regulation of mitochondrial biogenesis. The level of mtDNA in skeletal muscle of low-RFI lambs was higher than those ranked as high-RFI. Higher mRNA level of PGC-1α was also detected in low-RFI lambs in compared with the high-RFI group. However, no significant difference was observed between the RFI groups for mRNA expression of SIRT1. The results showed a negative relationship between phenotypic expression of RFI with mitochondrial biogenesis and PGC-1α mRNA level in skeletal muscle of fat-tailed lambs. Overally, our results suggest that the increased mitochondrial dynamics has a potential to regulates bioenergetic efficiency and energy expenditure in low-RFI lambs.