Objective Astatine ( 211 At) is a promising alpha emitter as an alternative to iodine ( 131 I). We are preparing the first-in-human (FIH) clinical trial of targeted alpha therapy for differentiated thyroid cancer in consultation with Pharmaceuticals and Medical Devices Agency. Here, we performed an extended single-dose toxicity examination under a reliability standard, as a preclinical safety assessment of 211 AtNaAt to determine the FIH dose. Methods 211 AtNaAt solution was injected into normal 6-week-old mice (male ( n  = 50) and female ( n  = 50), body weight: male 33.2 ± 1.7 g, female 27.3 ± 1.5 g), which were then divided into four groups: 5 MBq/kg ( n  = 20), 20 MBq/kg ( n  = 20), 50 MBq/kg ( n  = 30), saline control ( n  = 30). The mice were followed up for 5 days (primary evaluation point for acute toxicity: n  = 80) or 14 days ( n  = 20: evaluation point for recovery) to monitor general condition and body weight change. At the end of the observation period, necropsy, blood test, organ weight measurement, and histopathological examination were performed. For body weight, blood test, and organ weight, statistical analyses were performed to compare data between the control and injected groups. Results No abnormal findings were observed in the general condition of mice. In the 50 MBq/kg group, males (days 3 and 5) showed a significant decrease in body weight compared with the control. However, necropsy did not differ significantly beyond the range of spontaneous lesions. In the blood test, males (50 MBq/kg) and females (50 MBq/kg) showed a decrease in white blood cell and platelet counts on day 5, and recovery on day 14. In the testis, a considerable weight decrease was observed on day 14 (50 MBq/kg), and multinucleated giant cells were observed in all mice, indicating a significant change related to the administration of 211 AtNaAt. Conclusions In the extended single-dose toxicity study of 211 AtNaAt, administration of high doses resulted in weight loss, transient bone marrow suppression, and pathological changes in the testis, which require consideration in the FIH clinical trial.