The DNA-damage process of the U251 cells after being treated with ¹²⁵I-NGF is observed to show the antitumor mechanism of ¹²⁵I-NGF. The cell cycle arrest and the Western Blotting analysis were adopted to display the inherent mechanism. The micronucleus test and the single cell gel electrophoresis methods were used to observe DNA-damage changes of the U251 cells. The mechanisms involve the activation of ATM and ATR pathways by ¹²⁵I-NGF, which perhaps explains some of its anticancer activities related with nuclear transportations. In a dose-dependent manner, ¹²⁵I-NGF reduces the Cyclin B1 expression protein level rather than mRNA level in human cell lines of glioma. In U251 cells, phosphorylated Chk1, Chk2 and Cdc25c protein levels are elevated, whereas p53 and p21 levels remain unchanged. In summary, this study demonstrates that ¹²⁵I-NGF plays a role in glioma cancer growth via DNA-damage, ATM and ATR activation mechanisms and multiple proteins involved in the process.