Many studies have revealed the ATM alterations involved in cancer development and progression. In order to elucidate ATM deficiency in advanced GC and its clinical significance, a total of 20 exons of ATM gene, including frequently reported variations, were screened in 40 advanced primary GC and matched normal tissues using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing analysis. Furthermore, ATM mRNA level was analyzed using Real-time RT-PCR and in situ hybridization, and protein expression and phosphorylation at Ser1981 were measured by immunohistochemical assessment in tissue microarray of GC. Five variants were identified in 6 of 40 cases (15%), but no hot spot of variation was detected. However, decreased expression and phosphorylation of ATM were consistently presented in tumors. In a cohort of 70 GC samples, low level of phosphorylated ATM was significantly correlated with poor differentiation, lymph node metastasis and poor 5-year survival (P<0.05). These results indicated that ATM phosphorylation status might be a prognostic marker for individual therapy in advanced GC patients.