Background and purpose Simultaneous targeting of β1 integrin receptor and epidermal growth factor receptor (EGFR) showed higher level of radiosensitization in head and neck cancers than monotherapies. As EGFR inhibition is similarly performed in colorectal cancer (CRC), we investigated the radiosensitizing and anti-invasive potential of β1-integrin/EGFR inhibition in CRC cell lines grown in more physiological three-dimensional (3D) matrix-based cell cultures. Materials and methods DLD-1 and HT-29 cells were used for 3D-colony formation, invasion and proliferation assays and Western blotting. β1 integrin, focal adhesion kinase and EGFR were inhibited by AIIB2, TAE226 and Cetuximab, respectively. KRAS and BRAF knockdown were accomplished using small-interfering RNA technology. Single doses of X-rays ranged from 2 Gy to 6 Gy and 5-fluorouracil (5-FU) concentration was 10 μM. Results Neither β1-integrin/EGFR inhibition nor KRAS or BRAF depletion nor 5-FU significantly modified CRC cell radiosensitivity. Cetuximab, AIIB2 and Cetuximab/AIIB2 differentially modulated MAPK, JNK and AKT phosphorylation. AIIB2 and TAE226 significantly decreased cell invasion. Conclusions Our data show inefficiency of Cetuximab and AIIB2 on top of radiochemotherapy. The functions of KRAS and BRAF in therapy resistance remain unanswered and warrant further preclinical molecular-driven investigations. One promising approach might be β1 integrin targeting for reducing metastatic CRC cell spread.