Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP(2)) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P(2) but not PI(4)P(1) or PIP(3), releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP(2) in the plasma membrane and eliminated EGF-induced calpain activation. This PIP(2)-binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP(2) in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP(2) concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-gamma hydrolyzes PIP(2) in the protruding lamellipodia.