The oncogenic tyrosine kinase Src has a role in cancer development, especially by promoting invasive and metastatic behavior. It is, however, unclear which of the Srcregulated signaling cascades induce malignant phenotype in three-dimensional environment. One of Src substrates is ezrin, a cytoskeletal organiser and regulator of signal transduction. Ezrin expression correlates with poor outcome of diverse cancers and is essential in experimental metastatic osteosarcoma. We reconstituted genetically ezrin-deficient cells with wild-type (WT) or phosphorylation-deficient Y477F ezrin together with constitutively active Src. In two-dimensional cultures, Src induced malignant features regardless of the presence or absence of ezrin. In contrast, only WT ezrin-expressing cells grew efficiently in soft agar or in suspension. In Matrigel, only WT ezrin significantly promoted growth and invasion, and was targeted to specific regions on the plasma membrane. WT and Y477F ezrin-expressing cells showed marked differences only when growing or scattering in threedimensional matrix. Additional experiments showed that Y477-phosphorylated ezrin is also needed for the growth of Src-transformed epithelial cells in three-dimensional matrix. Cells lacking functional ezrin had reduced cyclin D levels and fewer cells in G2+S phase, possibly as a consequence of abnormal mTOR signaling, as ezrin Y477F cells showed lower expression of phosphorylated intermediates downstream of mTOR than WT cells. We conclude that the pathways activated by Src depend on the type of environment and that ezrin is a crucial element of Src-induced malignant features in cells growing inside three-dimensional environment.