Cell motility is a critical event in many processes and is underlined by complex signalling interactions. Although many components have been implicated in different forms of cell migration, identification of early key mediators of these events has proved difficult. One potential signalling intermediate, PLCγ1, has previously been implicated in growth-factor-mediated chemotaxis but its position and roles in more-complex motility events remain poorly understood. This study links PLCγ1 to early, integrin-regulated changes leading to cell motility. The key role of PLCγ1 was supported by findings that specific depletion of PLCγ1 by small interfering (si)RNA, or by pharmacological inhibition, or the absence of this isoform in PLCγ1–/– cells resulted in the failure to form cell protrusions and undergo cell spreading and elongation in response to integrin engagement. This integrin-PLCγ1 pathway was shown to underlie motility processes involved in morphogenesis of endothelial cells on basement membranes and invasion of cancer cells into such three-dimensional matrices. By combining cellular and biochemical approaches, we have further characterized this signalling pathway. Upstream of PLCγ1 activity, β1 integrin and Src kinase are demonstrated to be essential for phosphorylation of PLCγ1, formation of protein complexes and accumulation of intracellular calcium. Cancer cell invasion and the early morphological changes associated with cell motility were abolished by inhibition of β1 integrin or Src. Our findings establish PLCγ1 as a key player in integrin-mediated cell motility processes and identify other critical components of the signalling pathway involved in establishing a motile phenotype. This suggests a more general role for PLCγ1 in cell motility, functioning as a mediator of both growth factor and integrin-initiated signals.