Early diagnosis of cancer is a key factor for the success of treatment. For this reason, identification of highly sensitive and specific novel tumor markers is urgently needed. In the present study, the CM5 polyclonal antibody (CM5 pAb) raised against p53 of mouse origin was used to identify p53 structurally related protein(s) that may also play an important role in promoting or preventing lung cancer. Western blot analysis was performed on tumor tissues and corresponding normal tissues obtained from lung cancer patients. CM5 pAb reacted with a human protein with an apparent molecular weight of 90 kDa in the lung tumor tissue. The levels of this protein were greatly increased in 35 of the 37 (94.6%) lung tumor samples assessed, with only minimal expression in the normal adjacent tissues. The 90-kDa protein was immunoprecipitated by CM5 pAb and was subsequently identified by LC-MS/MS to be glucosidase II, a key protein involved in the quality control mechanism of glycoprotein folding. An investigation of the response to genotoxic stress and endoplasmic reticulum (ER) stress using A549 human lung adenocarcinoma cells demonstrated that glucosidase II exhibited a similar pattern of response as the p53 tumor suppressor. Protein levels of both p53 and glucosidase II were increased in response to UV irradiation but decreased in response to tunicamycin-induced ER stress. In conclusion, we demonstrated that a polyclonal antibody raised against mouse p53 could cross-react with human glucosidase II, which was found to be frequently overexpressed in human lung tumor tissues and exhibited a stress response similar to p53. The high frequency of glucosidase II overexpression, which to the best of our knowledge has not been previously described, indicates its crucial roles in lung tumorigenesis and is thus a valuable biomarker for facilitating the screening and/or diagnosis of lung cancer. However, further investigations concerning its relationship to p53 and its roles in ER and genotoxic stress are warranted.