The hypothalamus–pituitary–adrenal (HPA) axis is activated in most, but not all multiple sclerosis (MS) patients and is implicated in disease progression and comorbid mood disorders. In this post-mortem study, we investigated how HPA axis activity in MS is related to disease severity, neurodegeneration, depression, lesion pathology and gene expression in normal-appearing white matter (NAWM). In 42 MS patients, HPA axis activity was determined by measuring cortisol in cerebrospinal fluid (CSF) and counting hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons. Degree of neurodegeneration was based on levels of glutamate, tau and neurofilament in CSF. Duration of MS and time to EDSS 6 served as indicators of disease severity. Glutamate levels correlated with numbers of CRH-expressing neurons, most prominently in primary progressive MS patients, suggesting that neurodegeneration is a strong determinant of HPA axis activity. High cortisol levels were associated with slower disease progression, especially in females with secondary progressive MS. Patients with low cortisol levels had greater numbers of active lesions and tended towards having less remyelinated plaques than patients with high cortisol levels. Interestingly, NAWM of patients with high cortisol levels displayed elevated expression of glucocorticoid-responsive genes, such as CD163, and decreased expression of pro-inflammatory genes, such as tumor necrosis factor-α. Thus, HPA axis hyperactivity in MS coincides with low inflammation and/or high neurodegeneration, and may impact on lesion pathology and molecular mechanisms in NAWM and thereby be of great importance for suppression of disease activity.