RhoH is an atypical small G protein with defective GTPase activity that is specifically expressed in hematopoietic lineage cells. RhoH has been implicated in regulation of several physiological processes including hematopoiesis, integrin activation, and T cell differentiation and activation. In the present study, we investigated the role of RhoH in mast cells by generating RhoH knockout mice. Despite observing normal development of mast cells in vivo, passive systemic anaphylaxis and histamine release were impaired in these mice. We also observed defective degranulation and cytokine production upon FcεRI ligation in RhoH-deficient bone marrow-derived mast cells. Furthermore, FcεRI-dependent activation of Syk and phosphorylation of its downstream targets, including LAT, SLP76, PLCγ1, and PLCγ2 were impaired, however phosphorylation of the γ-subunit of FcεRI remained intact. We also found RhoH-Syk association that was greatly enhanced by active Fyn. Our results indicate that RhoH regulates FcεRI signaling in mast cells by facilitating Syk activation, possibly as an adaptor molecule for Syk.