Purpose: Integrin-mediated adhesion to extracellular matrix (ECM) contributes to the regulation of the cellular radiation response in various tumor entities. To evaluate whether targeting of β1 integrin enhances the radiosensitivity of head and neck SCC cell lines (HNSCC) was assessed using either inhibitory anti-β1 integrin antibodies or specific β1 integrin small interfering RNA (siRNA). Materials and methods: The HNSCC cell lines FaDu, UTSCC15 and UTSCC14 were used. Upon β1 integrin inhibition, colony formation, proliferation, DNA double strand breaks, adhesion, and migration as well as protein expression and phosphorylation of integrin downstream targets like Focal Adhesion Kinase and AKT were determined. Results: We found that siRNA- and antibody-mediated targeting of β1 integrin result in a dose- and cell line-dependent radiosensitization that was accompanied by a decreased cell proliferation and an increased number of radiogenic DNA double strand breaks. Analysis of signal transduction events revealed a dephosphorylation of focal adhesion proteins, prevention of radiation-induced phosphorylation of pro-survival protein kinases and impaired cell adhesion and migration upon blocking of β1 integrins. Conclusions: Our data suggest that β1 integrin critically contributes to the cellular radioresistance of HNSCC. Further studies are warranted to evaluate whether targeting β1 integrin emerges as novel approach to improve radiotherapy patients' outcome.