Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) promotes their desensitization and internalization. Here, we sought to determine the role of GRK2 on FcεRI signaling and mediator release in mast cells. The strategies utilized included lentiviral shRNA-mediated GRK2 knockdown, GRK2 gene deletion (GRK2flox/flox/cre recombinase) and overexpression of GRK2 and its regulator of G protein signaling homology (RH) domain (GRK2-RH). We found that silencing GRK2 expression caused ∼50% decrease in antigen-induced Ca2+ mobilization and degranulation but resulted in ablation of cytokine (IL-6 and IL-13) generation. The effect of GRK2 on cytokine generation does not require its catalytic activity but is mediated via the phosphorylation of p38 and Akt. Overexpression of GRK2 or its RH domain (GRK2-RH) enhanced antigen-induced mast cell degranulation and cytokine generation without affecting the expression levels of any of the FcεRI subunits (α, β, and γ). GRK2 or GRK2-RH had no effect on antigen-induced phosphorylation of FcεRIγ or Src but enhanced tyrosine phosphorylation of Syk. These data demonstrate that GRK2 modulates FcεRI signaling in mast cells via at leasttwomechanisms. OneinvolvesGRK2-RHand modulates tyrosine phosphorylation of Syk, and the other is mediated via the phosphorylation of p38 and Akt. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.