Cetuximab, a specific anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is used in cancer treatment. Although development of resistance to cetuximab is well recognized, the underlying mechanisms remain unclear. In the present study, we characterized cetuximab-resistant oral squamous cell carcinoma (OSCC) cell lines. The human OSCC cell lines HSC3, HSC4 and SAS were used in the present study. Effects of inhibitors including cetuximab on growth in cells were assessed by MTT assays. Southern blotting and immunofluorescence analysis were performed to examine protein expression and localization. Sphere formation was used to characterize stem cell-like properties. Floating aggregation culture was used for anchorage-independent growth. Cetuximab inhibited proliferation of HSC3 and HSC4 cells, but not SAS cells. Proliferation of all three cell lines was inhibited by the EGFR/ErbB2/ErbB4 inhibitor II. The EGFR inhibitor AG1478 strongly inhibited HSC3 and HSC4 proliferation, but that of SAS cells only moderately. EGFR proteins were localized on cell surface and phosphorylated in all three cell lines. SAS cells could proliferate in serum-free monolayer culture and formed spheres from single cells in floating culture. HSC3 and HSC4 could not proliferate under serum-free culture conditions and could not form spheres. Growth of SAS spheres required serum, and was inhibited by both AG1478 and cetuximab. Thus, cetuximab-resistant SAS cells not only engaged in EGFR-independent growth but also exhibited stem cell-like properties. However, growth was EGFR-dependent in aggregation culture, and the SAS cell aggregates became cetuximab-sensitive. This suggests that cetuximab sensitivity is not only cell-type-dependent but is also affected by the growth microenvironment.