VEGF is a key angiogenic factor that regulates proliferation and migration of endothelial cells via phosphorylation of ERK1/2 and p38, respectively. Here, we demonstrate that VEGF strongly induces the transcription of two dual specificity phosphatase (DUSP) genes, DUSP1 and DUSP5, in endothelial cells. Using fluorescence microscopy, FLIM and FCCS, we found that DUSP1/MKP-1 was localised both in the nucleus and cytoplasm of endothelial cells, where it existed in complex with p38 (KD (eff) 294 and 197 nM, respectively), whereas DUSP5 was localised in the nucleus of endothelial cells in complex with ERK1/2 (KD (eff) 345 nM). VEGF administration affected differentially the KD (eff) of the DUSP1/p38 and DUSP5/ERK1/2 complexes. Gain-of-function and lack-of-function approaches revealed that DUSP1/MKP-1 dephosphorylates primarily VEGF-phosphorylated p38, thereby inhibiting endothelial cell migration, whereas DUSP5 dephosphorylates VEGF-phosphorylated ERK1/2 inhibiting proliferation of endothelial cells. Moreover, DUSP5 exhibited considerable nuclear anchoring activity on ERK1/2 in the nucleus, thereby diminishing ERK1/2 export to the cytoplasm decreasing its further availability for activation.