Muskoxen (Ovibos moschatus), a taxonomically unique Arctic species, are increasingly exposed to climate and other anthropogenic changes. It is critical to develop and validate reliable tools to monitor their physiological stress response in order to assess the impacts of these changes. Here, we measured fecal glucocorticoid metabolite (FGM) levels in response to the administration of adrenocorticotropic hormone (ACTH) in the winter (1 IU/kg) and summer (2 IU/kg) using two enzyme immunoassays, one targeting primarily cortisol and the other targeting primarily corticosterone. Fecal cortisol levels varied substantially within and among individuals, and none of the animals in either challenge showed an increase in fecal cortisol following the injection of ACTH. By contrast, two of six (winter) and two of five (summer) muskoxen showed a clear response in fecal corticosterone levels (i.e., maximal percentage increase as compared to time 0 levels > 100%). Increases in fecal corticosterone post-ACTH injection occurred earlier and were of shorter duration in the summer than in the winter and fecal corticosterone levels were, in general, lower during the summer. These seasonal differences in FGM responses may be related to the use of different individuals (i.e., influence of sex, age, social status, etc.) and to seasonal variations in the metabolism and excretion of glucocorticoids, intestinal transit time, voluntary food intake, and fecal output and moisture content. Results from this study support using FGMs as a biomarker of hypothalamic-pituitary-adrenal axis activity in muskoxen, advance our understanding of the physiological adaptations of mammals living in highly seasonal and extreme environments such as the Arctic, and emphasize the importance of considering seasonality in other species when interpreting FGM levels.