Astatine, the heaviest member of the halogen group, is not only an analogous to iodine but also is a radionuclide that produce alpha particles. In recent decades, radioastatine(²¹¹At) has been concerned as a radiopharmaceutical due to the cytotoxic effect with high linear energy transfer (LET) and short particle path length. Although there are lots of remarkable clinical trials using ²¹¹At treatment, the mechanism of action after ²¹¹At administration is not fully understood. In this study, we reported that the cytotoxic effect of ²¹¹At in various cancer cells and a single-dose toxicity examination of ²¹¹At in mouse. Treatment of ²¹¹At led to induce cell growth inhibition in cancer cells including lung cancer cells (A549), prostate cancer cells (PC-3) and rat pancreatic cancer cells (AR42J) by dose and time dependent manner. However, human breast cancer cells (MDA-MB-231) and pancreatic cancer cells (BxPC-3) had relatively resistance to cell growth inhibition by using ²¹¹At. Interestingly, all the human cancer cells followed by ²¹¹At were dramatically increased cell growth inhibition in the long-term cytotoxic test by using colony forming assay. We estimated that DNA double strand breaks (DSBs) might induce cell growth inhibition, consequentially we found the activation of Poly (ADP-ribose) Polymerase-1 (PARP-1), a prominent DSBs marker, in human cancer cells followed by ²¹¹At. To observe the toxicity in mouse according to the ²¹¹At concentration, ²¹¹At was intravenously injected by various concentrations (0, 200, 300 kBq/g) and survival curves were estimated using Kaplan-Meier analysis. The 300 kBq/g group showed lower survival rate (P = 0.003) compared to control group (0 kBq/g), but 200 kBq/g group was not statistically significant. These results are expected to provide useful basic information for toxic prediction which might contribute to combination use of other anticancer drugs and the development of therapeutic radiopharmaceuticals.