Hydroxysafflor yellow A (HSYA) is the main segment of the safflower yellow pigments, and its second clinical study has been approved by the China SFDA for the treatment of brain blood vessel disease. Present studies were conducted to demonstrate the subchronic toxicity of HSYA. Sprague-Dawley (SD) rats were tested by daily intraperitoneal (IP) injection of HSYA at the doses of 180, 60, 20 mg/kg for 90 days. HSYA at high dose of 180 mg/kg and at middle dose of 60 mg/kg (90-day daily injection) induced a prolonged blood coagulation time without influencing the normal blood coagulation process. No animal died from hemorrhaging. The prolonged blood coagulation time was recovered to a normal level on the 28th day after withdrawing the drug. Kidney injury, including round tubular figures and a breaking-off of the tubular epithelium in histological slices under the microscope scenic, was only observed in the rats given by HSYA at 180 mg/kg. HSYA at dose of 180 mg/kg also increases the liver index without an obvious pathological change in liver histological analysis. There was no other organ injury found in this study. In conclusion, 90 days of HSYA treatment at a dose of 180 mg/kg, not at a dose of 60 mg/kg, induces a slight nephrotoxicity.