IgE receptor (FceRI) activation in mast cells triggers the release of inflammatory and allergic mediators from cytoplasmic granules. FceRI activation increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) which is accomplished by Ca²⁺ release from intracellular stores and the entry of Ca²⁺ through Ca²⁺-release activated Ca²⁺ (CRAC) channels. Mast cell activation leads to disassembly of the cortical F-actin cytoskeleton allowing fusion of cytoplasmic granules with the plasma membrane. Ca²⁺ mobilization is essential for cortical F-actin disassembly. Involvement of CRAC channels and mechanisms linking [Ca²⁺]_i increase to cortical F-actin reorganization remained, however, elusive. In the present study [Ca²⁺]_i was measured by fluorescent spectrometry, actin polymerization dynamics with biochemical and immunofluorescent techniques and degranulation through the release of b-hexosaminidase in mouse bone marrow-derived mast cells. As a result inhibition of CRAC channels with two different blockers, 2-APB or SKF-96365, blunted the increase in [Ca²⁺]_i and prevented the disassembly of the cortical F-actin and degranulation following mast cell activation. It is further shown that mast cells express the Ca²⁺-sensitive actin-binding protein villin, which acquires actin severing activity upon increase in [Ca²⁺]_i. Villin was colocalized with cortical F-actin in untreated mast cells. The colocalization of actin and villin was lost upon mast cell stimulation through FceRI, leading to cortical F-actin depolymerisation and villin redistribution towards the cell centre. However, when CRAC channels were blocked with 2-APB or SKF-96365 the colocalization of cortical F-actin and villin remained intact. Thus, villin may function as the Ca²⁺ sensor that mediates actin cytoskeleton rearrangements upon mast cell activation.