The aim of the current study was to investigate the presence of human papillomavirus (HPV) and evaluate its association with Ki-67 and cyclooxygenase-2 (COX-2) expressions in keratocystic odontogenic tumor (KCOT). Nineteen cases were included in the present study. Conventional PCR method and immunohistochemical analysis were performed for the detection of HPV-DNA and HPV-L1 capsid protein. Moreover, the expressions of Ki-67 and COX-2 proteins were analyzed immunohistochemically. HPV-DNA was detected in 36.8 % (7/19) of tumor samples, whilst HPV-L1 protein was identified in 68.4 % (13/19) of them. The Kappa coefficient statistical test showed a moderate agreement (kappa 0.424) between PCR and IHC assays for HPV detection. Expression of HPV-DNA was positively correlated with Ki-67 and COX-2 expressions (p < 0.05), whereas HPV-L1 positive staining was positively correlated with COX-2 (p < 0.05) and highly associated with those of Ki-67 (p < 0.01). There was no significant correlation between the presence of HPV and the recurrence of the studied lesions. The results of the current study showed that active HPV infection was present in the odontogenic epithelium of KCOT, and it was associated with increased proliferation rate and COX-2 expression. These findings suggest that HPV may have a role in the pathogenesis and aggressiveness of KCOT. Based on these conclusions, we recommend further investigations of HPV vaccine or antiviral therapy and COX-2 inhibitors as nonsurgical options in the prevention and management of KCOT.