<jats:title>Received June 8, 2022; accepted October 11, 2022</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Recent advances in omics techniques have allowed detailed genetic characterization of cortisol-producing adrenal adenoma (CPA). In contrast, the pathophysiology of CPAs has not been elucidated in detail on the level of tumor metabolic alterations.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>The current study conducted a comprehensive mass spectrometry imaging (MSI) map of CPAs in relation to clinical phenotypes and immunohistochemical profiles of steroidogenic enzymes. The study cohort comprised 46 patients with adrenal tumors including CPAs (n 35) and nonfunctional adenomas (n 11).</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Severity of cortisol hypersecretion was significantly correlated with 29 metabolites (adjusted P 0.05). Adrenal androgens derived from the classic androgen pathway were inversely correlated with both cortisol secretion (rs 0.41, adjusted P 0.035) and CYP11B1 expression (rs 0.77, adjusted P 2.00E-08). The extent of cortisol excess and tumor CYP11B1 expression further correlated with serotonin (rs 0.48 and 0.62, adjusted P 0.008 and 2.41E-05). Tumor size was found to be correlated with abundance of 13 fatty acids (adjusted P 0.05) and negatively associated with 9 polyunsaturated fatty acids including phosphatidic acid 38:8 (rs 0.56, adjusted P 0.009).</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>MSI reveals novel metabolic links between endocrine function and tumorigenesis, which will further support the understanding of CPA pathophysiology.</jats:p> </jats:sec>