The physiological role of brown adipose tissue (BAT) is quite different from that of white adipose tissue (WAT) by dissipating energy as heat instead of generating ATP. Mitochondrion is an essential organelle that plays a crucial role in energy production and cellular homeostasis. To gain a better understanding of energy metabolism in BAT, mitochondria were isolated and a systematic analysis of protein patterns was performed in WAT and BAT of rat with two-dimensional gel electrophoresis. Differentially expressed proteins were identified using mass spectrometry and it was found that the proteins involved in lipid and amino acid metabolisms, TCA cycle, and respiratory chain are more abundant in BAT than in WAT, which are further up-regulated in response to cold exposure in BAT. Furthermore, the study reveals, for the first time, a subset of COQ genes required for Coenzyme Q biosynthesis are significantly up-regulated after cold exposure in BAT. These findings suggest that Coenzyme Q increase is implicated in the non-shivering thermogenesis and contribute new data for understanding the unique process of energy metabolism in BAT.