A significant body of knowledge about radiobiology is based on studies of single dose cellular irradiation, despite the fact that conventional clinical applications using dose fractionation. In addition, cellular radiation response strongly depends on cell–cell and cell–extracellular matrix (ECM) interactions, which are poorly established in cancer cells grown under standard 2D cell culture conditions. In this study, we investigated the response of human colorectal carcinoma (CRC) DLD1 and HT29 cell lines, bearing distinct p53 mutations, to a single 2 or 10 Gy dose or fractionated 5 × 2 Gy doses of radiation using global transcriptomics analysis. To examine cellular response to radiation in a cell–ECM-interaction-dependent manner, CRC cells were grown under laminin-rich ECM 3D cell culture conditions. Microarray data analysis revealed that, overall, a total of 1,573 and 935 genes were differentially expressed (fold change >1.5; P < 0.05) in DLD1 and HT29 cells, respectively, at 4 h postirradiation. However, compared to a single dose of radiation, fractionated doses resulted in significantly different transcriptomic response in both CRC cell lines. Furthermore, pathway enrichment analysis indicated that p53 pathway and cell cycle/DNA damage repair or immune response functional categories were most significantly altered in DLD1 or HT29 cells, respectively, after fractionated irradiations. Novel observations of radiation-response-mediated activation of pro-survival pathways in CRC cells grown under lr-ECM 3D cell culture conditions using fractionated doses provide new directions for the development of more efficient radiotherapy strategies. Our results also indicated that cell line specific radiation response with or without activation of the conventional p53 pathway is ECM dependent, suggesting that the ECM is a key component in cellular radiation response.