Incidence of Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) has increased in recent years. HPV-associated OPSCC follows a distinct carcinogenic pathway from the HPV negative OPSCC. The molecular basis of disease progression due to HPV remains to be elucidated. Since clinic observation has revealed frequent and early lymph node metastasis and unusual distant metastasis in HPV-associated OPSCC as compared with HPV negative OPSCC, we would like to study whether β-catenin, one of the major proteins in the Wnt pathway and in regulation of epithelial-mesenchymal transition (EMT), is involved in HPV-associated OPSCC metastasis. The study samples (n = 209) were collected from formalin-fixed and paraffin-embedded or fine-needle aspiration OPSCC tissues. Expressions of p16 as a surrogate marker for HPV-associated cancer and β-catenin in these samples were determined by immunohistochemistry (IHC) analyses. Positive expression was defined as greater than 70% of tumor cells showing immunoreactivity for p16. Membrane expression of β-catenin was scored as a weight index (WI) = intensity (0, 1+, 2+, and 3+) x positive staining (%), where the membrane intensity on normal epithelium served as an internal positive control (3+). Nuclear staining of β-catenin was quantified as either positive or negative. Our results showed that p16 expression in OPSCC was significantly correlated with males (p<0.001), Stage III and IV (p = 0.009), undifferentiated phenotype (p=0.05), and positive lymph node (p<0.001). Membrane WI of β-catenin was inversely correlated with p16 positive OPSCC (p < 0.001) and positive lymph nodes (p = 0.035), while nuclear staining of β-catenin was associated with p16 positive OPSCC (p = 0.007). Low level of membrane β-catenin (cut-off by the median value) was significantly associated with disease free survival and overall survival (p < 0.05 in both cases). Our study suggested that cytoplasmic and nuclear localization of β-catenin is associated with p16 and therefore, HPV status and lymph node metastasis. Molecular study on the regulation of β-catenin by HPV oncogenes E6 or E7 and the role of β-catenin in metastasis of OPSCC is ongoing in our laboratory. (This study was supported by US National Institutes of Health grant HHSN2610201000125C and Georgia Cancer Coalition Distinguished Cancer Scholar Award to GZC).