Histamine H4 receptor (H4R) has emerged as a potential therapeutic target for pruritus and inflammatory skin disorders. In this study, we sought to identify novel H4R antagonists and evaluate their potential as antipruritic agents. High-throughput screening of 60,480 small-molecule compounds identified three novel H4R antagonists, among which H4Ri-A01 showed the highest potency with an IC50 value of 355 nM. H4Ri-A01 exhibited high selectivity, showing no significant effects on H1R, H2R, 5-HT2A receptor, PAR2, TGR5, or MRGPRX2 activity at 10 μM. Molecular docking analysis predicted that H4Ri-A01 binds to the H4R ligand-binding pocket occupied by the reference antagonist JNJ7777120 and has a slightly higher predicted binding affinity. H4Ri-A01 showed no detectable cytotoxicity in CHO-K1 or HEK293T cells at concentrations up to 10 μM. In LAD-2 human mast cells, H4Ri-A01 concentration-dependently inhibited degranulation induced by histamine and the selective H4R agonist 4-methylhistamine, demonstrating functional inhibition of H4R-mediated mast cell responses. Furthermore, H4Ri-A01 significantly and dose-dependently reduced scratching behavior in a histamine-induced mouse model of pruritus. Collectively, these findings demonstrate that H4Ri-A01 is a potent and selective novel H4R antagonist with cellular and in vivo antipruritic activity. H4Ri-A01 therefore represents a promising lead compound for the development of H4R-targeted therapeutics for chronic pruritus.