Cardiolipin (CL) is a unique mitochondrial phospholipid that, in skeletal muscle, is enriched with linoleic acid (18:2n6). Together, CL content and CL 18:2n6 composition are critical determinants of mitochondrial function. Skeletal muscle is comprised of slow and fast fibers that have high and low mitochondrial content, respectively. In response to overloading and unloading stimuli, these muscles undergo a fast-to-slow oxidative fiber type shift and a slow-to-fast glycolytic fiber type shift, respectively, with a concomitant change in mitochondrial content. Here, we examined changes in CL content and CL 18:2n6 composition under these conditions along with tafazzin (Taz) protein, which is a transacylase enzyme that generates CL lipids enriched with 18:2n6. Our results show that CL content, CL 18:2n6 composition, and Taz protein content increased with an overload stimulus in plantaris. Conversely, CL content and CL 18:2n6 composition was reduced with an unloaded stimulus in soleus. Interestingly, Taz protein was increased in the unloaded soleus, suggesting that Taz may provide some form of compensation for decreased CL content and CL 18:2n6 composition. Together, this study highlights the dynamic nature of CL and Taz in skeletal muscle, and future studies will examine the physiological significance behind the changes in CL content, CL 18:2n6 and Taz.