Objectives: This study was performed to analyze the toxicity and to find the lethal dose of the test substance Water-soluble Carthami-flos pharmacopuncture (WCF) when used as a single intravenous-dose in 6-week-old, male and female Sprague-Dawley rats. Methods: The experiment was conducted at Biotoxtech according to Good Laboratory Practices. 20 female and 20 male Spague-Dawley rats were divided into 4 groups of 5 female and 5 male animals per group. The rats in the three experimental groups received single intravenous injections with 0.125-mL, 0.25-mL and 0.5-mL/animal doses of WCF, Groups 2, 3, and 4, respectively, and the control group, Group 1, received a single intravenous injection with a 0.5-mL dose of normal saline. Clinical signs were observed and body weight measurements were carried out for 14 days following the injections. At the end of the observation period, hematology, clinical chemistry, histopathological tests and necropsy were performed on the injected parts. Results: No deaths occurred in any of the groups. Also, no significant changes in body weight, hematological parameters or clinical chemistry test results between the control group and the experimental groups were observed. Visual inspection after necropsy showed no abnormalities. Histopathological tests on the injected parts showed no significant differences, except for Group 1 females; however, the result was spontaneous generation and had no toxicological meaning because it was not dose-dependent. Therefore, this study showed that WCF had no effect on the injected parts in terms of clinical signs, body weight, hematology, clinical chemistry, and necropsy. Conclusion: As a result of single intravenous-dose tests of the test substance WCF in 4 groups of rats, the lethal dose for both males and females exceeded 0.5 mL/animal. Therefore, WCF is a relatively safe pharmacopuncture that can be used for treatment, but further studies should be performed.