Peimine, is derived from the Fritillaria genus, has long been recognized for its anti-inflammatory activity. In this study, we investigated peimine\'s potential as an antipruritic and anti-inflammatory agent using a combination of in vitro, ex vivo, and in vivo techniques. The results of in vitro calcium imaging indicated that Peimine inhibited several pruritus-related receptors, including TRPM3, MRGPRX2, and MRGPRB2, with minimal or no effect on TRPA1, TRPV1, and TRPV4. Ex vivo experiments in dorsal root ganglion (DRG) neurons confirmed significant inhibition of TRPM3 activity. β-hexosaminidase assays further demonstrated that peimine suppressed MRGPRB2 on peritoneal mast cells (PMCs) and MRGPRX2 on Human Mast Cells 1.2 (HMC 1.2) cells mediated degranulation in mast cells. In vivo, behavioral models of acute itch showed that peimine effectively reduced scratching behaviors induced by pregnenolone sulfate (TRPM3 agonist) and compound 48/80 (MRGPRB2 agonist). Additionally, in a compound 48/80 induced paw edema model, peimine attenuated inflammatory swelling, supporting its anti-inflammatory potential. Collectively, these findings highlight peimine as a promising therapeutic candidate for alleviating pruritus.