Oncogenic transformation often leads to the disruption of the actin cytoskeleton. Activation of the classical Ras-Raf-MEK1/2-ERK1/2 signalling cascade has been implicated in the effects of oncogenes such as Ras and Src on the cytoskeleton. Many of the studies of the effects of oncogenes on the cytoskeleton have made use of chemical inhibitors of MEK1/2 but it is now clear that these inhibitors also inactivate MEK5 in the MEK5-ERK5 MAP kinase pathway raising the possibility that this pathway may also be involved in oncogenic transformation. We therefore investigated whether activation of ERK5 can lead to disruption of the actin cytoskeleton. We show that activation of ERK5 can lead to loss of actin stress fibres, but by a distinct mechanism to ERK1/2. We demonstrate that ERK5 is activated by oncogenic Src as demonstrated by translocation of endogenous ERK5 from the cytoplasm to nucleus and activation of an ERK5-dependent transcriptional reporter and that ERK5 activation is required for Src-mediated transformation. We also show that in Src-transformed cells inhibition of ERK1/2 signalling is not sufficient for reappearance of the actin cytoskeleton and that ERK5 activation contributes to cytoskeletal disruption by Src. Our results suggest that multiple MAP kinase pathways downstream of oncogenes participate in cytoskeletal alterations.