Objective: BRAF activating mutations represents approximately 10% colorectal cancer (CRC) patients who are associated with a poor prognosis in stage II, III and IV. Therefore, to better understand the antitumor effects of Selumetinib in BRAF V600E -mutated CRC, gene set enrichment analysis (GSEA) of gene expression profile of BRAFV 600E - mutated HT-29 cells treated of Selumetinib is proposed in this study.Methods: The differentially expressed genes across the comparison of groups of non- treated and treated of Selumetinib in BRAF V600E -mutated HT-29 cells were analyzed by GSEA. Results: We identified the significant pathways of DNA dependent DNA replication, DNA replication, regulation of DNA replication, cell activation, leukocyte activation, receptor complex, endoplasmic reticulum membrane, RNA processing, RRNA processing, amino acid transmembrane transporter activity, nucleolar part, ribonucleoprotein complex, ribosome biogenesis and assembly and regulation of protein modification process for the upregulated genes and the significant pathways of response to hypoxia, response to extracellular stimulus, ATPase coupled to transmembrane movement of ions, ATPase coupled to transmembrane movement of ions phosphorylative mechanism, lipase activity, MAP kinase activity, phospholipase activity and actin filament for the downregulated genes relevant to resistance of Selumetinib in BRAF V600E - mutated HT-29 cells. Our data revealed that GLI2, IL8, LAT2, ICOSLG, TLR4, SPINK5, HDAC4, LAT and TGFB1 are the upregulated important genes and PDIA2, TGFB2, GIPR and GCGR are the importantly downregulated genes associated with resistance of BRAF V600E -mutated HT-29 cells to Selumetinib. Conclusion: We identified the upregulated and/or downregulated genes significantly associated with the resistance of Selumetinib in BRAF V600E -mutated HT-29 cells. These top upregulated and/or downregulated genes have significant value in prediction of sensitivity of CRC patients to Selumetinib and in identification of effective combination strategy for treatment of CRC patients.