Endoscopic ultrasonography-guided fine-needle aspiration biopsy (EUS-FNAB) has been shown to be a highly accurate technique for distinguishing benign from malignant pancreatic masses. In this study, we examined p53 immunohistochemical analysis in FNAB specimens obtained from solid pancreatic diseases, and prospectively evaluated clinical applications for the diagnosis of malignancy in combination routine histological examination. Tissue specimens obtained from 62 pancreatic masses (51 pancreatic cancers and 11 chronic pancreatitis) by EUS-FNAB were evaluated by routine histological examination and p53 immunostaining. The conventional EUS-FNA diagnostic test statistics for the pancreatic masses were as follows: 76% sensitivity, 91% specificity and 79% accuracy. p53 protein overexpression was observed in 67% patients with pancreatic cancer, but not in patients with chronic pancreatitis. If the diagnosis of malignancy was made using the combination of p53 protein overexpression and conventional histological examination, the diagnostic test statistics changed as follows: 90% sensitivity, 91% specificity and 92% accuracy. p53 immunostaining in combination with routine histological examination of EUS-FNAB may improve the diagnostic accuracy for pancreatic cancer.