Photodynamic therapy (PDT) is a locally administered therapy currently being investigated in various clinical and preclinical settings. Tumor-host interaction is an important determinant of tumor biology and response to treatments. Here we report for the first time the effects of PDT on an orthotopic, murine mammary tumor model. PDT utilizes two individually nontoxic components: (a) the localization in the target site of a photosensitizing drug; and (b) the activation of the photosensitizer by light of an appropriate wavelength and energy. PDT after a single dose of the photosensitizer MV6401 induced drug dose-dependent, long-term blood flow shut down and tumor growth delay in the MCaIV tumor, grown in the mammary fat pad. The plasma half-life of MV6401 was approximately 20 min, and the drug was confined to the vascular compartment shortly after administration. However, it accumulated in the interstitial compartment at 2-6 h after the administration. Two equal MV6401 doses injected 4 h and 15 min before the light administration allowed the photosensitizer to localize in both vascular and tumor cell compartments. The fractionated drug dose PDT more effectively induced tumor growth delay than the same total dose given as a single dose either at 4 h or at 15 min before light administration. The long-term effect of the fractionated drug PDT on blood flow was also more extensive than single-dose PDT. Fractionated photosensitizer dosing PDT offers a new strategy to optimize PDT therapy.