Abstract Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by different enzyme deficiencies in the steroid hormone synthesis leading to a disturbed cortisol biosynthesis. The medical treatment of CAH includes suboptimal ACTH-suppressing high glucocorticoid doses to reduce adrenal hyperplasia and overproduction of androgens. These inappropriate corticoid substitutions are often associated with undesirable side effects such as arterial hypertension, growth failure and obesity. Since the current therapy of patients with CAH is often unsatisfactory, innovative treatment options are required. The aim of our study was to specifically block the melanocortin 2 receptor (MC2R) signaling pathway as an alternative treatment strategy for CAH. We tested ACTH-derived selective peptides with incorporation of various synthetic non-natural amino acids in the activation motif of ACTH. To study the antagonistic activity of the peptides, cAMP production of MC2R/MRAP stably transfected human embryonic kidney (HEK) 293 cells were measured. All new synthesized peptide antagonists reduced ACTH-stimulated MC2R activity as competitive inhibitors indicated by a reduced in vitro cAMP response. Cells pre-incubated with peptide LNP009 showed the most efficient blockade of the MC2R and the highest shift of EC50 of ACTH (33.8 nM ± 0.08 vs. 7.3 nM ± 0.09). LNP009 was additionally tested for specificity concerning the other known melanocortin receptors and showed no antagonistic effect up to 1 μM on MC3, MC4 or MC5 receptor transiently transfected HEK 293 cells. To further investigate the inhibitory effect of our most potent antagonist peptide LNP009 on the steroid hormone response, we assessed steroidogenic enzyme expression of the human adrenocortical tumor cell line NCI-H295RA and performed mass spectrometry analyzes of steroids in the cell culture supernatant. Pre-incubation with LNP009 reduced the expression of the genes CYP21A2, CYP11B1 and HSD3B2 in NCI-H295RA cells and significantly reduced the synthesis of aldosterone (P=0.046; n=3), cortisol (P=0.020; n=3) and corticosterone (P=0.035; n=3). With the successful blocking of the ACTH binding and signal transduction by our antagonistic peptides, we anticipate an alternative approach for optimizing the treatment of CAH patients lacking the side effects of the currently used ACTH-suppressing corticoid therapy.