Prion protein (PrP) is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that functions as a unique pathogenic agent in transmissible spongiform encephalopathy (TSE). In the past decade, overexpression of PrP was observed in a number of human malignant tumors, such as gastric, breast and pancreatic cancer. However, the role of PrP expression in squamous cell carcinoma is rarely documented. To screen PrP expression in head and neck squamous cell carcinoma (HNSCCs), the paraffin-embedded specimens of 92 pathologically diagnosed HNSCCs were assessed by PrP-specific immunohistochemistry (IHC). A total of 55.43% (51/92) of the tested carcinoma tissues were PrP-positive. The rate of positivity and the staining intensity of PrP were closely related with the pathological degree of the HNSCCs; a higher rate of PrP expression was noted in the group of poorly differentiated cancers. PrP-positivity rates increased along with the progression of the clinical grade of the carcinomas. Further evaluation of the associations between PrP expression and the data concerning p53 abnormalities and human papillomavirus (HPV) infection in these samples as previously described, revealed that PrP-positive staining was more frequently detected in the tissues with p53-positive accumulation and the wild-type TP53 gene. The patients with a proline (Pro) polymorphism in SNP72 of TP53 showed significantly higher PrP-positive rates than those with arginine (Arg). No notable difference in PrP expression was identified between the HPV-positive and HPV-negative group. These data indicate a close association of PrP expression with clinical and histological differentiation of HNSCCs, as well as abnormalities of p53.