Mast cells (MCs) are important in allergic reactions. Here, we assess the anti-allergic effects of the anti-cancer drug tozasertib specifically regarding regulatory effects on MCs activation.,Tozasertib effects on MC degranulation was determined by measuring β-hexosaminidase and histamine release and by assessing morphological changes in RBL-2H3 and mouse bone marrow-derived mast cells (BMMCs) stimulated with mouse anti-dinitrophenyl (DNP)-immunoglobulin E (IgE)/DNP-human serum albumin (HSA) or human LAD2 cells activated with phorbol-12-myristate 13-acetate plus calcium ionophore (PMACI). Western blots were performed to detect the expression of molecules involved in NF-κB, MAPK, and aurora kinase signaling. In vivo anti-allergic effects of tozasertib were determined in the murine IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin-induced active systemic anaphylaxis (ASA) models.,Tozasertib treatment resulted in significantly decreased high-affinity IgE receptor (FcεRI) or PMACI-mediated degranulation in RBL-2H3 cells and in BMMCs or LAD2 cells as evidenced by β-hexosaminidase or histamine levels. Similarly, tozasertib prevented morphological changes in MCs, such as particle release and F-actin reorganization. In addition, tozasertib diminished the expression levels of phosphorylated (p)-NF-κB p65, p-Erk1/2, p-p38 and p-Aurora A/B markedly, indicating that tozasertib can inhibit the signaling pathway mediating MC activation. Tozasertib attenuated IgE/Ag-induced PCA dose-dependently, as evidenced by reduced Evans blue staining. Similarly, tozasertib reduced body temperature levels and serum histamine levels in OVA-challenged ASA mice.,The aurora kinase inhibitor tozasertib suppressed MC activation in vitro and in vivo. Tozasertib may be a potential drug for targeting MC activation to treat allergic diseases or mastocytosis.,This article is protected by copyright. All rights reserved.