Purpose　Tween-80　is　one　of　the　most　important　causes　resulting　in　anaphylactoid　reaction.　However,　its　mechanism　remains　unclear.　Proteomic　characterizations　of　mast　cells＇　excreta　in　response　to　Tween-80　are　assayed　to　investigate　the　mechanism　of　anaphylactoid　reaction.　Experimental　design　A　label-free　LCMS/MS-based　proteomics　is　used　to　analyze　Tween-80-stimulated　Laboratory　of　Allergic　Diseases　2　(LAD2)　mast　cells　releasates.　The　results　of　proteomic　are　analyzed　by　bioinformatics　analysis.　Western　blotting　is　used　to　verify　the　expression　of　proteins.　Results　Overall,　endocytosis,　nuclear　factor　kappa-light-chain-enhancer　of　activated　B　cells　(NF-kappa　B),　and　calcium　signaling　pathways　play　important　roles　in　Tween-80-induced　LAD2　cells　activation　by　bioinformatics　analysis.　The　expressions　of　relative　proteins　including　actin-related　protein　2/3　complexes,　vacuolar　protein　sorting-associated　protein,　phosphorylation　of　transcription　factor　of　P105　and　P65,　phosphorylation　of　inositol　1,4,5-trisphosphate　receptor　(IP3R),　phosphoinositide　phospholipase　C　gamma　(PLC　gamma),　and　protein　kinase　C　(PKC),　are　significantly　increased　in　Tween-80　group　compared　to　control.　Tween-80　might　be　internalized　via　endocytosis,　which　induces　degranulation　by　PLC　gamma/PKC　pathways　mediated　calcium　influx,　and　promotes　the　generation　of　inflammatory　mediators　via　NF-kappa　B　pathway　resulting　in　anaphylactoid　reaction.