Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) are great tools to monitor the activity changes of signaling molecules in living cells. Recently, we and others have succeeded to generate transgenic mice stably-expressing such FRET biosensors and to visualize the activity changes of signaling molecule in living mice. Here, by in vivo two-photon excitation microscopy with such FRET mice, the activity of protein kinase A (PKA) examined in intratumoral vascular endothelial cells. We found that the level of PKA activity was significantly lower in the intratumor endothelial cells than the subcutaneous endothelial cells. PKA activation with a cAMP analog alleviated the tumor vascular hyperpermeability, suggesting that the low PKA activity in the endothelial cells may be responsible for the tumor-tissue hyperpermeability. In addition, Motesanib, a kinase inhibitor for vascular endothelial growth factor (VEGF) receptor, activated tumor endothelial PKA and reduced the vascular permeability in the tumor. Notably, in cultured human umbilical vascular endothelial cells, VEGF activated PKA rather than decreasing its activity, highlighting the remarkable difference between its actions in vitro and in vivo.