​Atopic dermatitis is a chronic inflammatory skin disease characterized by severe itching. Recently, non-histaminergic itch pathways such as Mas-related G Protein-coupled receptor (MRGPR) have been considered important mediators. This study aimed to examine the effects of Peptide X on MRGPRX1 and MRGPRX2, receptors associated with non-histaminergic itch. HEK293T cells were transfected with human MRGPRX1 or MRGPRX2, and calcium imaging was used to investigate receptor activity after Peptide X treatment. Peptide X activated both MRGPRX1 and MRGPRX2 in a concentration-dependent. Especially, when 200μM Peptide X was treated in MRGPRX2, the response level significantly increased compared to the control group. This suggests that Peptide X may act as a direct agonist or positive modulator of this receptor. Our results demonstrate that Peptide X activates MRGPRX1 and MRGPRX2, providing new insights into the non-histaminergic itch pathway in atopic dermatitis. Also, Peptide X shows that it may be a useful agent for studying MRGPR-mediated signaling pathways.