In vitro studies revealed that insulin resistance might be associated with the intracellular formation of ceramide, the second messenger in the sphingomyelin signaling pathway. The aim of the present study was to examine the content and composition of fatty acids in ceramide and sphingomyelin in human muscle and to evaluate their relationships with insulin sensitivity. The study was conducted on 27 male subjects with normal glucose tolerance. Euglycemic-hyperinsulinemic clamps and biopsies of vastus lateralis muscle were performed. In 10 subjects, additional biopsies were taken after a 4-h clamp and after a clamp with concurrent Intralipid/heparin infusion. We identified 13 ceramides and sphingomyelins according to fatty acid residues. Insulin sensitivity was related to total ceramide content (r = −0.49, P = 0.01) and to ceramide consisting of palmitic (r = −0.48, P = 0.011), palmitoleic (r = −0.45, P = 0.019), mirystic (r = −0.42, P = 0.028), and nervonic acid (r = −0.39, P = 0.047). Hyperinsulinemia did not affect estimated muscle parameters. Intralipid/heparin infusion resulted in a 24.73% decrease in insulin sensitivity (P = 0.007) and a 47.81% increase in ceramide content (P = 0.005). These changes were significantly related to each other (r = −0.64, P = 0.046). A relationship with the decrease in insulin sensitivity was also observed for ceramides consisting of palmitic (r = −0.68, P = 0.03) and linoleic (r = −0.66, P = 0.038) acid. Our data indicate that the sphingomyelin signaling pathway in muscle might be an important factor determining the development of insulin resistance in humans.