Phosphatidylserine (PS) is asymmetrically distributed across the plasma membrane, located predominantly on the inner leaflet in healthy cells. Translocation of PS to the outer leaflet makes it available as a target for biological therapies. We examined PS translocation after radiosurgery in an animal model of brain arteriovenous malformation (AVM). An arteriovenous fistula was created by end-to-side anastomosis of the left external jugular vein to the common carotid artery in 6-week-old, male Sprague Dawley rats. Six weeks after AVM creation, 15 rats underwent Gamma Knife stereotactic radiosurgery receiving a single 15 Gy dose to the margin of the fistula; 15 rats received sham treatment. Externalization of PS was examined by intravenous injection of a PS-specific near-infrared probe, PSVue-794, and in vivo fluorescence optical imaging at 1, 7, 21, 42, 63 and 84 days postirradiation. Fluorescent signaling indicative of PS translocation to the luminal cell surface accumulated in the AVM region, in both irradiated and nonirradiated animals, at all time points. Fluorescence was localized specifically to the AVM region and was not present in any other anatomical sites. Translocated PS increased over time in irradiated rats (P < 0.001) but not in sham-irradiated rats and this difference reached statistical significance at day 84 (P < 0.05). In summary, vessels within the mature rat AVM demonstrate elevated PS externalization compared to normal vessels. A single dose of ionizing radiation can increase PS externalization in a time-dependent manner. Strict localization of PS externalization within the AVM region suggests that stereotactic radiosurgery can serve as an effective priming agent and PS may be a suitable candidate for vascular-targeting approaches to AVM treatment.