We hypothesized that chronic inhibition of epidermal growth factor receptor (EGFR) by cetuximab, a monoclonal anti-EGFR antibody, induces up-regulation of its ligands resulting in resistance and that microRNAs (miRs) play an important role in the ligand regulation in head and neck squamous cell carcinoma (HNSCC). Genome-wide changes in gene and miR expression were determined in cetuximab-sensitive cell line, SCC1, and its resistant derivative 1Cc8 using DNA microarrays and RT-PCR. The effects of differentially expressed EGFR ligands and miRs were examined by MTS, colony formation, ELISA, and western blot assays. Heparin-binding EGF-like growth factor (HB-EGF) and its regulator, miR-212, were differentially expressed with statistical significance when SCC1 and 1Cc8 were compared for gene and miR expression. Stimulation with HB-EGF induced cetuximab resistance in sensitive cell lines. Inhibition of HB-EGF and the addition of miR-212 mimic induced cetuximab sensitivity in resistant cell lines. MicroRNA-212 and HB-EGF expression were inversely correlated in an additional 33 HNSCC and keratinocyte cell lines. Six tumors and 46 plasma samples from HNSCC patients were examined for HB-EGF levels. HB-EGF plasma levels were lower in newly diagnosed HNSCC patients when compared to patients with recurrent disease. Increased expression of HB-EGF due to down-regulation of miR-212 is a possible mechanism of cetuximab resistance. The combination of EGFR ligand inhibitors or miR modulators with cetuximab may improve the clinical outcome of cetuximab therapy in HNSCC.