During amphibian gastrulation, mesodermal cell movements depend on both cell–cell and cell–matrix interactions. Ectodermal cells from the blastocoel roof use α5β1 integrins to assemble a fibronectin-rich extracellular matrix on which mesodermal cells migrate using the same α5β1 integrin. In this report, we show that the tyrosine phosphatase xPTP-PESTr can prevent fibronectin fibril formation when overexpressed in ectodermal cells resulting in delayed gastrulation. In addition, isolated ectodermal cells overexpressing xPTP-PESTr are able to spread on fibronectin using the α5β1 integrin in the absence of activin-A induction and before the onset of gastrulation. We further show that while the inhibition of fibrillogenesis depends on the phosphatase activity of xPTP-PESTr, induction of cell spreading does not. Finally, while cell spreading is usually associated with cellmigration, xPTP-PESTr promotes ectodermal cell spreading on fibronectin but also reduces cellmigration in response to activin-A, suggesting an adverse effect on cell translocation. We propose that xPTP-PESTr overexpression adversely affectcellmigration by preventing de-adhesion of cells from the substrate.