Background: Topical tacrolimus has been widely used in the treatment of inflammatory and immune dermatoses for its immunosuppression effect. However, a transient irritation like itching, burning induced by tacrolimus is common when initial application, which is similar to pseudo-allergic reaction. MAS-related G protein-coupled receptor-X2 (MRGPRX2) in mast cells (MCs) mediates drug-induced pseudo-allergic reaction and IgE-independent pruritis in chronic skin diseases. Whether MRGPRX2 participates in above tacrolimus adverse reaction should be addressed. Further, immunosuppression mechanism of tacrolimus on MCs is greatly ignored. Methods: Wild-type (WT) mice, kit w-sh/w-sh mice and MrgprB2 deficient (MUT) mice were applied to explore the mechanism of initial irritant reaction and immunosuppression of tacrolimus on the skin MrgprB2 in MCs in vivo. LAD2 cells and MRGPRX2-knockdown LAD2 cells were used to confirm the regulation of MRGPRX2 by tacrolimus in vitro. Results: Tacrolimus could trigger IgE-independent dermatitis when initial application through MrgprB2-related MCs degranulation. Using FK-DNS, a fluorescently labeled tacrolimus, we found tacrolimus could bind to MRGPRX2 directly. Interestingly, after long-term tacrolimus treatment, the initial itching and inflammatory reaction faded away without IgE change. Hence, longstanding treatment with tacrolimus suppressed MRGPRX2/B2 expression and decreased inflammatory cytokines release. Conclusion: Our study provides for the first time a novel target for tacrolimus, demonstrating that short-term tacrolimus treatment induces pseudo-allergic reaction via MRGPRX2/B2 in MCs, while long-term treatment dampens MRGPRX2/B2 expression, leading to decreased inflammatory cytokines release and immune cells recruitment, which may contribute to its potent immunosuppression effect in the treatment of inflammatory and immune skin diseases.