Targeted Alpha Therapy (TAT) using Astatine-211 (At-211) is a promising radiopharmaceutical approach due to its chemical similarity to iodine which allows for cellular uptake via the sodium/iodide symporter (NIS). While it is established that At-211 typically enters cells through the NIS, its specific toxicological profile across various tissues remains unclear. To address this, the present study established a histopathological baseline for At-211 through in vitro and in vivo assessments. In vitro, CCK-8 and colony-forming assays revealed that prostate cancer cell lines (PC3) exhibited significantly higher sensitivity to At-211 at lower doses than other tissues. Notably, immunoblotting and uptake studies suggested that this toxicity may occur through NIS-independent mechanisms. In vivo, male ICR mice intravenously administered At-211 (30.5, 61, and 122 kBq/g) showed dose-dependent toxicity—including apoptosis and hyperplasia—specifically in prostate tissues at 3 and 4 weeks post-injection. These findings confirm the selective toxicity of At-211 toward the prostate, providing the fundamental toxicological data necessary for the future development of At-211-based radiopharmaceuticals.