BACKGROUND: Psychotic major depression (PMD) differs from non-psychotic MD (NPMD) in psychopathology and is linked to changes in brain volumetry and hypothalamic-pituitary-adrenal (HPA) axis function that can be reflected by its principal output - the glucocorticoid cortisol. NPMD patients exhibit smaller hippocampi than healthy controls (HC), purportedly representing exposure to chronic stress. However, the relationship between the individual clinical phenotype, hippocampal volume and diurnal cortisol signaling remains unclear. METHODS: Since understanding the interplay among symptoms, neuroimaging and HPA function is crucial for discerning biological differences between PMD and NPMD, this study explored the link between clinical phenotype, hippocampal structural MRI and circadian plasma cortisol levels in 32 HC, 27 NPMD and 26 PMD patients. RESULTS: PMD patients showed significantly elevated evening (6 p.m. - 1 a.m.) cortisol levels compared to NPMD and HC, while NPMD and HC did not differ. No group differences in hippocampal volume were observed, but a significant interaction effect emerged between overnight (1 a.m. - 9 a.m.) cortisol levels, hippocampal volume, and clinical phenotype. NPMD patients displayed a negative correlation between overnight cortisol levels and hippocampal volume, which was specific to the ascending cortisol curve (2 a.m. - 5 a.m.) and absent in PMD and HC. The hippocampus-cortisol interaction was associated with depressive symptom severity in NPMD but not PMD, where cortisol alone predicted greater severity. CONCLUSIONS: These findings imply a time-dependent relationship between hippocampal volume and overnight cortisol in NPMD, which is absent in PMD and HC. In contrast, PMD patients exhibited increased evening cortisol levels. In an exploratory analysis, these effects were also related to symptom severity at similar timepoints. While correlational, these results point to distinct neurobiological mechanisms underlying NPMD and PMD, which are potentially related to the heterogeneous clinical manifestations.