We report a novel approach to selectively close single blood vessels within tissue using multiphoton absorption-based photothermolysis (multiphoton photothermolysis) without the need of exogenous agents. The treatment process is monitored by in vivo reflectance confocal microscopy in real time. Closure of single targeted vessels of varying sizes ranging from capillaries to venules was demonstrated. We also demonstrated that deeply situated blood vessels could be closed precisely while preserving adjacent overlying superficial blood vessels. In vivo confocal Raman spectroscopy of the treatment sites confirmed vessel closure as being mediated by local coagulative damage. Partial vessel occlusion could be achieved, and it is accompanied by increased intravascular blood cell speed. Multiphoton photothermolysis under real-time reflectance confocal imaging guidance provides a novel precision medicine approach for noninvasive, precise microsurgery treatment of vascular diseases on a per-vessel/per-lesion basis. The method could also be used for building ischemic stroke models for basic biology study.