Factors contained in physiological microenvironments in tissues where mast cells differentiate and reside may influence mast cell responsiveness and modify antigen-dependent activation. A possible direct or indirect role of mast cell responses in diabetes mellitus prompted us to study the impact of insulin treatment on antigen triggered signaling pathways downstream of FcepsilonRI in bone marrow-derived mouse mast cells (BMMCs). We found that insulin alone stimulates tyrosine phosphorylation of tyrosine kinases Lyn, Syk, Fyn, the adapter protein Gab2 (Grb2-associated binding protein 2), Akt and activates ERK, JNK and p38 kinase. Effect of insulin on FcepsilonRI signaling pathways was marked by enhanced phosphorylation of Lyn, Fyn, Gab2 and Akt. Furthermore, BMMC stimulated with antigen in the presence of insulin responded with enhanced protein kinase theta (PKCtheta) activity and increased JNK phosphorylation when compared to BMMC triggered with antigen alone. Functional studies reveal enhanced degranulation and altered cytoskeletal rearrangement when BMMCs were treated simultaneously with insulin and antigen. Our results suggest that insulin tunes antigen-mediated responses of mast cells.