Nestin, an intermediate filament protein and marker of undifferentiated cells, is expressed in several cancers. Nestin is important for neuronal survival and is a regulator of myogenesis but its function in malignancy is ambiguous. We show that nestin downregulation leads to a redistribution of phosphorylated focal adhesion kinase (pFAK, also known as PTK2) to focal adhesions and alterations in focal adhesion turnover. Nestin downregulation also leads to an increase in the protein levels of integrin α5β1 at the cell membrane, activation of integrin β1 and an increase in integrin clustering. These effects have striking consequences for cell invasion, as nestin downregulation leads to a significant increase in pFAK- and integrin-dependent matrix degradation and cell invasion. Our results indicate that nestin regulates the localisation and functions of FAK and integrin. Because nestin has been shown to be prevalent in a number of specific cancers, our observations have broad ramifications for the roles of nestin in malignant transformation.