Increased production and activation of matrix metalloproteinase-2 (MMP-2) are critical events in skeletal muscle angiogenesis and are known to occur in response to mechanical stresses. We hypothesized that reorganization of the actin cytoskeleton would increase endothelial cell production and activation of MMP-2 and that this increase would require a MAPK dependent signalling pathway in endothelial cells. The pharmacological actin depolymerization agent Cytochalasin D (CytoD), increased expression of MMP-2 and membrane type 1 (MT1)-MMP mRNA, and this was reduced significantly in the presence of the JNK inhibitor SP600125. Activation of JNK by anisomycin was sufficient to induce expression of both MMP-2 and MT1-MMP mRNA in quiescent cells. Downregulation of c-Jun, a downstream target of JNK, with small interference (si)RNA inhibited MMP-2 expression in response to anisomycin. Inhibition of PI3K, but not JNK, significantly decreased the amount of active MMP-2 following CytoD stimulation with a concurrent decrease in MT1-MMP protein. Physiological reorganization of actin occurs during VEGF stimulation. VEGF induced MMP-2 protein production and activation, as well as MT1-MMP protein production, depended on PI3K activity. VEGF induced MMP-2 mRNA expression was reduced by inhibition of JNK or by treatment with c-Jun siRNA. In summary, our results provide novel insight into the signalling cascades initiated in the early stages of angiogenesis through the reorganization of the actin cytoskeleton and demonstrate a critical role for JNK in regulating MMP-2 and MT1-MMP mRNA expression while PI3K regulates protein levels of both MMP-2 and MT1-MMP.