Intestinal mucosal restitution occurs by epithelial cell migration, rather than proliferation, to reseal superficial wounds after injury. Polyamines are essential for stimulation of intestinal epithelial cell (IEC) migration during restitution in association with their ability to regulate Ca²⁺ homeostasis, but the exact mechanism by which polyamines induce cytosolic free Ca²⁺ ([Ca²⁺]_cyt) concentration remains unclear. Phospholipase-C1 (PLC-1) catalyzes the formation of inositol-1,4,5-trisphosphate (IP₃) that is implicated in the regulation of [Ca²⁺]_cyt by modulating Ca²⁺ store mobilization and Ca²⁺ influx. The current study tested the hypothesis that polyamines are involved in PLC-1 activity regulating [Ca²⁺]_cyt and cell migration after wounding. Depletion of cellular polyamines by -difluoromethylornithine (DFMO) inhibited PLC-1 expression in differentiated IECs (stable Cdx2-transfected IEC-6 cells) as indicated by substantial decreases in levels of PLC-1 mRNA and protein, and its enzyme product IP₃. Polyamine-deficient cells also displayed decreased [Ca²⁺]_cyt and inhibited cell migration. Decreased levels of PLC-1 by treatment with U-73122 or transfection with siRNA specifically targeting PLC-1 (siPLC-1) also decreased IP3, reduced resting [Ca²⁺]_cyt and Ca²⁺ influx after store depletion, and suppressed cell migration in control cells. In contrast, stimulation of PLC-1 by 2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)-benzenesulfonamide (m-3M3FBS) induced IP₃, increased [Ca²⁺]_cyt, and promoted cell migration in polyamine-deficient cells. These results indicate that polyamines are absolutely required for PLC-1 expression in IECs and that polyamine-mediated PLC-1 signaling stimulates cell migration during restitution as a result of increased [Ca²⁺]_cyt.