AIM: To investigate the expression of Weel and phosphorylated Weel protein in colorectal cancer and normal tissues, and analyze the regulatory effect of Weel on cell proliferation in the human colorectal cancer cell line HCT116. METHODS: The expression of Wee1, p-Wee1 (Ser53) and p-Wee1 (Ser642) was detected by immunohistochemistry in colorectal cancer and normal tissues. The changes in Wee1, p-Wee1 (Ser53) and p-Wee1 (Ser642) expression were detected by Western blot in starved HCT116 cells. HCT116 cells were then cultured in medium containing different concentrations of Wee1 inhibitor PD407824. Cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay, and the morphology of the cells was observed by light microscopy. RESULTS: The positive rates of Wee1, p-Wee1 (ser53), and p-Wee1 (ser642) expression (68.00%, 85.00% and 91.00%) in colorectal cancer were significantly higher than those in normal tissues (9.62%, 21.15% and 42.31%) (P < 0.05 or P < 0.01). The expression of the Wee1 and its phosphorylated forms rose mainly at 6, 12 and 24 h. The proliferation of HCT116 cells was inhibited by Wee1 inhibitor PD407824. CONCLUSION: The levels of Wee1 and its phosphorylation forms closely relate to the proliferation of colorectal cancer cells. Wee1 inhibitor may be a potential new treatment for colorectal cancer in the future. © 2015 Baishideng Publishing Group Inc. All rights reserved.