Background: The clinical impact of the monoclonal antibody cetuximab targeting the EGFR in colorectal cancer (CRC) is widely recognized. Nevertheless, the onset of cetuximab resistance is a serious issue that limits the effectiveness of this drug in targeted therapies. Unraveling the molecular players involved in cancer resistance is the first step towards the identification of alternative signaling pathways that can be targeted to circumvent resistance mechanisms restoring the efficacy of therapeutic treatments in a tailored manner. Methods: By applying a nanoLC-MS/MS TMT isobaric labeling-based approach, we have delineated a molecular hallmark of cetuximab-resistance in CRC. Results: We identified macrophage migration inhibitory factor (MIF) as a molecular determinant capable of triggering cancer resistance in sensitive human CRC cells. Blocking the MIF axis in resistant cells by a selective MIF inhibitor restores cell sensitivity to cetuximab. The combined treatment with cetuximab and the MIF inhibitor further enhanced cell growth inhibition in CRC resistant cell lines with a synergistic effect depending on inhibition of key downstream effectors of the MAPK and AKT signaling pathways. Conclusions: Collectively, our results suggest the association of MIF signaling and its dysregulation to cetuximab drug resistance, paving the way to the development of personalized combination therapies targeting the MIF axis.