Background:Usually, chemoradiotherapy can be used for the treatment of a locally advanced colorectal cancer (CRC) before surgery. On the other hand, some studies have shown that fractional radiation of tumor cells leads to chemoresistance. The aim of this study was to evaluate the chemoresistance of radioresistant sub-line (RR sub-line).Methods: This study was done in Hamadan University of Medical Sciences in 2017-2018. MTT assay and sub-G1 fraction analysis by flow cytometry were used to evaluate cross-resistance of RR sub-line to Gefitinib and Regorafenib. Real-time PCR was used to investigate the role of four miRNAs and their target genes in cross-resistance of RR sub-line. The t test and repeated measures test were used for the assessment of statistical significance between groups.Results:The IC50 of Gefitinib and Regorafenib for RR sub-line were significantly higher than those of parental cell line. On the other hand, the resistance index of RR sub-line for Gefitinib and Regorafenib were 1.92 and 1.44, respectively. The sub-G1 fraction of RR sub-line following treatment with Gefitinib and Regorafenib was significantly lower than that of parental cell line (P=0.012 and P=0.038, respectively). The expression of miR-9, Let-7e, and Let-7b in RRsub-line was significantly lower than that of parental cell line. However, NRAS, IGF1R, NFKB1, and CCND1 found to be upregulated in RR sub-line in comparison with the parental cell line. Conclusion: We can conclude that acquired RR sub-line was cross-resistance to Gefitinib and Regorafenib. Furthermore, miR-9/NFKB1, let-7b/CCND1, let-7e/NRAS, and IGF1R played essential roles in chemoradioresistance of CRC.