Sphingolipids are important components of cell membranes that may also serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. The aim of this study was to examine the effect of 5 weeks of aerobic training on key enzymes and intermediates of ceramide metabolism in skeletal muscles. The experiments were carried out on rats divided into two groups: (1) sedentary and (2) trained for 5 weeks (on a treadmill). The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (nSMase and aSMase), neutral and alkaline ceramidases (nCDase and alCDase) and the content of sphingolipids was determined in three types of skeletal muscle. We also measured the fasting plasma insulin and glucose concentration for calculating HOMA-IR (homeostasis model assessment) for estimating insulin resistance. We found that the activities of aSMase and SPT increase in muscle in the trained group. These changes were followed by elevation in the content of sphinganine. The activities of both isoforms of ceramidase were reduced in muscle in the trained group. Although the activities of SPT and SMases increased and the activity of CDases decreased, the ceramide content did not change in any of the studied muscle. Although ceramide level did not change, we noticed increased insulin sensitivity in trained animals. It is concluded that training affects the activity of key enzymes of ceramide metabolism but also activates other metabolic pathways which affect ceramide metabolism in skeletal muscles.