The antivascular effects of photodynamic therapy (PDT) and their mechanisms are not clearly understood. Here, we examined the effects of PDT with a novel photosensitizer MV6401 on the microvasculature in a mammary tumor (MCaIV) grown in a murine dorsal skinfold chamber and in normal tissue controls. The mice were irradiated with light 15 min after i.v. administration of MV6401 when the drug was localized only in the vascular compartment, as shown by fluorescence microscopy and immunohistochemistry. PDT with MV6401 caused a dose-dependent biphasic blood flow stasis and vascular hyperpermeability, as determined by intravital microscopy. This biphasic response was classified into two components: (a) an acute response observed immediately after PDT; and (b) a long-term response observed at times greater than 3 h after PDT. The acute temporal vascular effects were characteristic of vasoconstriction but not of thrombus formation. However, the long-term vascular shutdown was mediated by thrombus formation, as evidenced by histological evaluation and inhibition with heparin. Minimal effects were observed in normal vessels after antivascular doses used against the tumor, but there was no long-term vascular damage. In concert with the stasis, a dose-dependent tumor growth delay was observed. This study provides mechanistic insights into antitumor vascular effects of PDT and suggests novel strategies for tumor treatment with PDT.