Cardiolipin (CL) is a phospholipid at the heart of mitochondrial metabolism, which plays a key role in mitochondrial function and bioenergetics. Among mitochondrial activity regulators, SIRT3 plays a crucial role in controlling the acetylation status of many enzymes participating in the energy metabolism in particular concerning lipid metabolism and fatty acid oxidation. Data suggest that possible connection may exist between SIRT3 and CL status that has not been evaluated in skeletal muscle. In the present study, we have characterized skeletal muscle lipids as well as mitochondrial lipids composition in mice overexpressing long (SIRT3-M1) and short (SIRT3-M3) isoforms of SIRT3. Particular attention has been paid for CL. We reported no alteration in muscle lipids content and fatty acids composition between the two mice SIRT3 strains and the control mice. However, mitochondrial CL content was significantly decreased in SIRT3-M3 mice and associated to an upregulation of tafazzin gene expression. In addition, mitochondrial phospholipids and fatty acids composition was altered with an increase in the PC/PE ratio and arachidonic acid content and a reduction in the MUFA/SFA ratio. These modifications in mitochondrial membrane composition are associated with a reduction in the enzymatic activities of mitochondrial respiratory chain complexes I and IV. In spite of these mitochondrial enzymatic alterations, skeletal muscle mitochondrial respiration remained similar in SIRT3-M3 and control mice. Surprisingly, none of those metabolic alterations were detected in mitochondria from SIRT3-M1 mice. In conclusion, our data indicate a specific action of the shorter SIRT3 isoform on lipid mitochondrial membrane biosynthesis and functioning.