ATF4 is a member of the cAMP-responsive element-binding protein family of basic zipper-containing proteins, a family of transcription factors phosphorylated at serines residues by protein kinase A. The family has been proved to be able to stimulate the transcription of the genes containing CRE elements. Elevated ATF4 expression was detected in some tumors including breast carcinoma compared to their corresponding nontumor tissues. p-ATF4 (ser 245), a phosphorylated form of ATF4 protein at serine 245 site, was believed to be an active type of this protein. However, its expression and clinical significance in malignant tumors including non-small cell lung cancer were not reported up to date. In the current study, we investigate the expression of p-ATF4 (ser 245) in non-small cell lung cancer using tissue microarray and immunohistochemistry. p-ATF4 (ser 245) immunostaining was detected in nucleus and cytoplasm in cancer cells and normal lung epithelial cells. Compared to bronchial epithelium and submucosal glands (total positive rate, 14.6% (12/82)), there was increased expression of p-ATF4 (ser 245) in non-small cell lung cancer cells (total positive rate, 42.7% (35/82)) (p < 0.05). In addition, increased expression of p-ATF4 (ser 245) was associated with lymph node metastasis and advanced TNM stages (III and IV) in non-small cell lung cancer (p < 0.05). Immunofluorescent staining confirmed nuclear and cytoplasmic expression of p-ATF4 (ser 245) in lung and cancer tissues, and also in non-small cell lung cancer cell lines including NCI-H157 and LTE cells. These results indicate that increased expression of p-ATF4 (ser 245) may contribute to cancer development of non-small cell lung cancer and may be a potential cancer marker.