The effects of long-term dexamethasone treatment on diaphragm muscle were studied in female and male rats. Compared with pair-fed control animals, dexamethasone treatment did not significantly affect estrous cycling or peak serum estradiol levels; however, testosterone levels were significantly increased in females and decreased in males. Dexamethasone significantly reduced body and costal diaphragm weights, but to a lesser extent in females than in males. Reductions in diaphragm weight were proportional to reductions in body weight. In females and males, dexamethasone treatment significantly decreased diaphragm fiber (types I and II) cross-sectional area and the relative expression of myosin heavy chain isoform 2B. With the exception of type I fiber atrophy, these changes occurred to a lesser extent in females. Dexamethasone did not significantly affect specific forces. Dexamethasone significantly increased twitch one-half relaxation time and fatigue resistance indexes in males but not in females. In conclusion, the effects of long-term dexamethasone treatment were gender specific, with significantly fewer effects in females, and changes in serum testosterone levels were associated with these findings.