Corticosterone (CORT), the major glucocorticoid in rodents, is secreted from the adrenal gland, affects various organs in the body and regulates energy metabolism as a stress response. Although local steroidogenesis of androgens and estrogens in skeletal muscles has been previously reported, local CORT synthesis in skeletal muscle remains unconfirmed. In the present study, we investigated steroidogenic activities in a clonal myoblastic cell line, C2C12 cells. Three enzymes involved in CORT synthesis, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450c21 and cytochrome P45011ß, were identified in C2C12 cells by detecting the enzymatic reaction products with LC–MS/MS analysis. Only one enzyme that mediates cholesterol cleavage was not detected in the cells. After the addition of pregnenolone-sulfate conjugates to the cell culture medium, pregnenolone was detected and increased according to the incubation time. In conclusion, CORT synthesis occurs in C2C12 cells, and it is suggested that the initial steroidogenesis substrate is the pregnenolone-sulfate conjugate.