p16 is a cyclin-dependent kinase inhibitor and a cardinal regulator of the cell cycle. The relationship between p16 overexpression and poor prognosis of breast cancer has been reported in some studies. This study aimed to evaluate p16 expression in breast cancer in comparison to normal breast tissue and determine the association between p16 expression and clinicopathological parameters in breast cancer. Paraffin blocks of 110 samples were studied. These included 40 invasive breast carcinoma (tumor group) and normal tissue adjacent to the tumor (tumor control), as well as 30 normal mammoplasty specimens (normal control). Samples were from the pathology archive of Alzahra Hospital, Isfahan, Iran, from 2016 to 2020. p16 expression was studied and compared in these three groups using the immunohistochemistry technique. Moreover, the relationship between p16 expression and age, tumor size, carcinoma subtype, tumor grade, and lymph node involvement was investigated in the tumor group. SPSS version 16 was used to analyze data. p16 expression showed a significant difference between the tumor group and the two control groups with a significantly higher expression in the tumor group. There was a significant direct relationship between the intensity of p16 expression and the number of involved lymph nodes (P < 0.001). No significant relationship was detected between p16 expression and other clinicopathological factors. p16 seems to have a rather significant expression in breast cancer in comparison to normal breast parenchyma. However, among clinicopathological parameters, we found only a direct relationship between lymph node involvement and intensity of p16 expression.