Following adherence of neutrophils to the endothelium, neutrophils undergo a major morphological change which is a necessary prelude to their extravasation. We show here that this shape change is triggered by an elevation of cytosolic IP3, to provoke physiological Ca2+ influx through a store-operated mechanism. This transition from a spherical to “flattened” neutrophil morphology is rapid (about 100 s) and is accompanied by an apparent rapid expansion of the area of the plasma membrane. However, no new membrane is added into the plasma membrane. Pharmacological inhibition of calpain-activation, which is triggered by Ca2+ influx during neutrophil spreading, prevents normal cell flattening. In calpain-suppressed cells, an aberrant form of cell spreading can occur where an uncoordinated and localised expansion of the plasma membrane is evident. These data show that rapid neutrophil spreading is triggered by Ca2+ influx which causes activation of calpain, and release of furled plasma membrane to allow its apparent “expansion”.