Dimethylsulfoxide (DMSO), a universal solvent, is frequently used to dissolve various classes of chemicals for the evaluation of their biological activities. In one such evaluation, we noticed that DMSO itself caused cellular proliferation and interfered with high affinity IgE receptor ($Fc{varepsilon}RI$)- mediated degranulation of mast cells. DMSO caused cellular proliferation of RBL-2H3 cells by phosphorylating both extracellular-signal regulated kinase (ERK) and $M_2$-type pyruvate kinase ($M_2PK$) through which the enzymatic activity of $M_2PK$ was reduced. Allergenic activation of $Fc{varepsilon}RI$ caused the tyrosine phosphorylations of signaling components of $Fc{varepsilon}RI$, such as Syk, $PLC{gamma}1$, $PLC{gamma}2$, ERK, and $M_2PK$. In these allergenic activated RBL-2H3 cells, DMSO specifically inhibited $Fc{varepsilon}RI$-mediated tyrosine phosphorylation of $M_2PK$, blocked $Fc{varepsilon}RI$-mediated inhibition of the enzymatic activity of $M_2PK$, and then inhibited $Fc{varepsilon}RI$-mediated degranulation. These results suggest that DMSO causes cellular proliferation and mast cell degranulation through differential modulation of $M_2PK$ in resting and allergenic activated cells.