Introduction. Although cetuximab has been widely used in the treatment of colon cancer, a large number of patients eventually develop drug resistance. Therefore, it is essential to clarify the mechanism of drug resistance. Methods. In this study, we combined in silico analysis and a single guide RNA (sgRNA) library to locate cetuximab‐sensitive genes. Cell proliferation, apoptosis, and cell cycle were assessed to validate the change in cetuximab sensitivity. Finally, western blotting was performed to detect changes in epidermal growth factor (EGFR) upstream and downstream genes. Results. Using in silico analysis and the sgRNA library, MEIS3 was confirmed as the cetuximab‐sensitive gene. Further experiments indicated that the expression of MEIS3 could determine the level of cetuximab. Meanwhile, MEIS3‐inhibited cells were sensitive to mesenchymal epithelial transition factor (c‐Met) and protein kinase B (Akt) inhibitors, which is related to the change in phosphorylation of c‐Met and degradation of Akt. Conclusion. MEIS3 modified the sensitivity to cetuximab through c‐Met and Akt.