Photodynamic therapy (PDT) has been shown to inactivate blood-borne pathogenic microorganisms in vitro. The method may be used to purify blood in the body, kill pathogenic microorganisms, and treat difficult diseases. Our aim was to investigate the safety of photodynamic blood purification (PBP) therapy using an in vivo blood circulation experiment in an animal model. Twenty-four New Zealand rabbits were used as experimental subjects; 12 received PDT and 12 served as negative controls. Extracorporeal blood bypass was established using the femoral artery and vein. A sterile disposable irradiation chamber was connected in the bypass pathway. Hematoporphyrin monomethyl ether was injected intravenously as a photosensitizer with an initial bolus of 3.7 mg, followed by a continuous infusion at 24 mg/h during PDT administration. Five minutes after initial injection, a laser beam was vertically focused on the irradiation chamber side wall, with a 9.5 cm2 spot area, 1 h exposure time, and 20 mW/cm2 power density. Six animals received a single PDT application, and six received PDT every other day for three applications. The 12 control group animals underwent extracorporeal blood circulation but did not receive the photosensitizer or light treatment. Blood samples were taken 20 min, 1 day, 4 days, and 7 days after PDT treatment for analysis of cell counts, coagulation, liver and renal function, and other biochemical changes. On the 7th day, animals were sacrificed, and parenchymal organs were evaluated for morphological changes. There were no significant differences in white blood cells, red blood cells, or destroyingplatelets after PDT compared with the control group. There was a little significant difference in alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, uric acid, blood urea nitrogen, and creatinine in the PDT group compared with the control group, except at individual time points. We found no significant damage in the heart, liver, spleen, lungs, kidneys, or other organs after PDT. This short-duration, fixed-strength PBP method did not cause changes in blood parameters or in the structure or function of major organs in an animal model. These findings suggest that PBP is safe in vivo and has potential as a new therapy for inactivating blood-borne microorganisms.