Mitochondria are functionally specialized in different tissues, and a detailed understanding of this specialization is important to elucidate mitochondrial involvement in normal physiology and disease. In adaptive thermogenesis, brown fat converts mitochondrial energy to heat, whereas tissue-specific functions of mitochondria in white fat are less characterized. Here we apply high-resolution quantitative mass spectrometry to directly and accurately compare the in vivo mouse mitochondrial proteomes of brown and white adipocytes. Their proteomes are substantially different qualitatively and quantitatively and are furthermore characterized by tissue-specific protein isoforms, which are modulated by cold exposure. At transcript and proteome levels, brown fat mitochondria are more similar to their counterparts in muscle. Conversely, white fat mitochondria not only selectively express proteins that support anabolic functions but also degrade xenobiotics, revealing a protective function of this tissue. In vivo comparison of organellar proteomes can thus directly address functional questions in metabolism.