The tumor-associated microenvironment has been shown to protect tumor cells from treatment, and the extracellular matrix (ECM) is known to affect drug resistance as a key regulator of the tumor microenvironment. However, little is known about cell adhesion-mediated drug resistance (CAM-DR) due to cell-ECM contact in patients with oral squamous cell carcinoma (OSCC). In the present study, we evaluated the ECM molecule fibronectin (FN) using DNA microarray data obtained from parental and 5-FU-resistant OSCC cell lines. We investigated the effects of cell adhesion to FN on 5-FU resistance in OSCC cells and examined the activation of FN receptor β1 integrin-mediated survival regulators such as ILK, Akt and NF-κB. In addition, we investigated whether FNIII14, a 22-mer peptide derived from FN that potently prevents β1 integrin-mediated adhesion to FN, could overcome CAM-DR against 5-FU in OSCC cells and examined the activation of survival regulators and apoptosis-related molecules. Consequently, we obtained the following results. FN was extracellularly overexpressed in the 5-FU-resistant cells compared with that observed in the 5-FU-sensitive cells. Cell adhesion to FN enhanced 5-FU resistance and activated integrin-mediated ILK/Akt/NF-κB survival signaling in the 5-FU-resistant OSCC cells. Furthermore, the inhibition of cell adhesion to FN by FNIII14 enhanced chemosensitivity to 5-FU and apoptosis by suppressing ILK/Akt/NF-κB signaling in the 5-FU-resistant cells. These novel findings demonstrate that FN is a potentially useful biomarker and therapeutic target for improving the treatment of OSCC, particularly in the setting of 5-FU resistance.