•Villin role in predicting tumor behavior in colorectal cancer patients are limited.•Villin expression related to tumor stage and tumor site.•Villin expression predicts aggressive histopathological findings and tumor behavior, and consequently poor cancer outcomes. Colorectal cancer is one of the most common cancers and the leading cause of cancer-related deaths worldwide. The incidence is gradually increasing, and the mortality and recurrence rates of the disease remain high. This study was conducted as a cross-sectional study using tissue samples of 106 patients who underwent surgery at Sina Hospital from 2021 to 2022. After histopathological examination and identification of the pathological features of the tumor, the samples were subjected to immunohistochemical staining using a monoclonal antibody against villin. In this study, we observed a significant association between villin expression and tumor depth, as well as a correlation between villin expression and tumor location (colon or rectum). However, no association was found between villin expression and the number of affected lymph nodes and age, sex, tumor grade, and size. Furthermore, there was no significant association between villin expression and tumor vascular or neural invasion. The extent of local invasion and metastasis are important factors in disease progression and can lead to treatment failure. Therefore, new biomarkers are needed to identify patients at risk of local and distant metastases and to enable appropriate treatment of patients.