Aggregation of the high affinity IgE receptor (FceRI) on mast cells (MCs) causes MC degranulation, a process that involves cortical F-actin disassembly. Actin depolymerization may be controlled by increase of cytosolic Ca²⁺. Entry of Ca²⁺ through the Ca²⁺-release activated Ca²⁺ (CRAC) channels is powerfully regulated by the serum- and glucocorticoid-inducible kinase SGK1. Moreover, SGK1-deficient (sgk1^-/-) MCs have a decreased FceRI -dependent degranulation. The present study addressed whether SGK1-dependent Ca²⁺ entry through CRAC channels is required for actin cytoskeleton rearrangements in MCs and whether defects in actin rearrangements could underlie decreased degranulation of sgk1^-/- MCs. Confirming previous results, sgk1^-/-mast cells had an impaired FceRI -dependent degranulation, as measured by the release of b-hexosaminidase. However, when CRAC channels were inhibited by 2-APB (50 mM), MC degranulation was strongly decreased in both sgk1^+/+ and sgk1^-/- cells and the difference between genotypes was abolished. Moreover, degranulation of both sgk1^+/+ and sgk1^-/- MCs was impaired by actin-stabilizing (phallacidin) and enhanced by actin-disrupting (cytochalasin) agents to the similar extent. Monomeric (G) versus filamentous (F) actin content was analyzed by FACS using DNase I Alexa-488, which recognizes G-actin, and phalloidin Alexa-647 detecting F-actin. Alternatively, G/F-actin ratio was determined by Western blot of detergent soluble and insoluble cell fractions, containing G- and F-actin, respectively. Both methods indicated that G/F-actin ratio increased in sgk1^+/+ but not insgk1^-/- mast cells upon FceRI ligation, reflecting actin depolymeraziation. This effect was abolished by 2-APB in sgk1^+/+ cells. Our studies identify a novel mechanism of actin cytoskeleton regulation through SGK1-dependent Ca²⁺ entry.