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 led to a redistribution of pFAK to focal adhesions (FA) and alterations in FA turnover. Nestin-downregulation also led to an increase in the cell membrane (CM) protein levels of integrin α5β1, activation of β1, and an increase in integrin clustering. These effects had striking consequences for cell invasion, as nestin-downregulation led to a significant increase in pFAK and integrin -dependent matrix degradation and cell invasion. Our results indicate that nestin regulates FAK and integrin localization and functions. Since 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.