This study was conducted to describe the modulation of plasma epidermal growth factor receptor (EGFR) ligands in EGFR-positive metastatic colorectal cancer (mCRC) patients during treatment with cetuximab and irinotecan and to explore the clinical implication of plasma levels' variations as potential biomarkers of benefit. Plasma amphiregulin (AR), epidermal growth factor (EGF), transforming growth factor-α, and heparin binding-EGF were assessed by ELISA in 45 chemorefractory mCRC patients, treated with cetuximab and irinotecan. Plasma levels were measured before and 1 h after the first administration of cetuximab, before and 1 h after the second administration, and before the third and the fifth cycles. KRAS and BRAF mutational status were determined. EGFR ligands' levels were differently modulated according to tumor KRAS and BRAF mutational status. In KRAS wild-type patients (n = 34), AR and EGF early increased and higher increases were significantly associated with worse clinical outcome. By adopting a specific cut-off value, patients with higher levels of AR 1 h after the first administration had significantly worse response rate, progression free survival, and overall survival. This hypothesis-generating study shows that EGFR ligands are significantly modulated by cetuximab plus irinotecan according to KRAS and BRAF mutational status, and they warrant further investigation as pharmacodynamic markers of resistance to anti-EGFRs.