ACTH-dependent hypercortisolism caused by a pituitary adenoma Cushing's disease (CD) is the most common cause of endogenous Cushing's syndrome. CD is often associated with several morbidities, including hypertension, diabetes, osteoporosis/bone fractures, secondary infections, and increased cardiovascular mortality. While the majority (≈80%) of the corticotrophinomas visible on pituitary magnetic resonance imaging are microadenomas (MICs, <10 mm of diameter), some tumors are macroadenomas (MACs, ≥10 mm) with increased growth potential and invasiveness, exceptionally exhibiting malignant demeanor. In addition, larger and invasive MACs are associated with a significant increased risk of local complications, such as hypopituitarism and visual defects. Given the clinical and molecular heterogeneity of corticotrophinomas, the aim of this study was to investigate the pattern of genetic differential expression between MIC and MAC, including the invasiveness grade as a criterion for categorizing these tumors. In this study, were included tumor samples from patients with clinical, laboratorial, radiological, and histopathological diagnosis of hypercortisolism due to an ACTH-producing pituitary adenoma. Differential gene expression was studied using an Affymetrix microarray platform in 12 corticotrophinomas, classified as non-invasive MIC (  = 4) and MAC (  = 5), and invasive MAC (  = 3), according to modified Hardy criteria. Somatic mutations in were also investigated, but none of the patients exhibited variants. Differential expression analysis demonstrated that non-invasive MIC and MAC have a similar genetic signature, while invasive MACs exhibited a differential expression profile. Among the genes differentially expressed, we highlighted , and , and their differential expression was validated through quantitative real-time PCR in another cohort of 15 non-invasive and 3 invasive cortocotrophinomas. We also identified potential biological pathways associated with growth and invasiveness, TGF-β and G protein signaling pathways, DNA damage response pathway, and pathways associated with focal adhesion. Our study revealed a differential pattern of genetic signature in a subgroup of MAC, supporting a genetic influence on corticotrophinomas in patients with CD.