Butyrate is a short-chain fatty acid decomposed from dietary fiber and has been shown to have effects on inhibition of proliferation but induction of apoptosis in colorectal cancer cells. However, clinical trials have yielded ambiguous outcomes with regard to its antitumor activities. In this study, we aimed to explore the molecular mechanisms underlying the sensitivity of colorectal cancer cells to sodium butyrate (NaB). RNA sequencing was used to establish the whole-transcriptome profile in NaB-treated versus untreated colorectal cancer cells. Differentially expressed genes were bioinformatically analyzed to predict their possible involvement in NaB-triggered cell death, and the expression of eight dysregulated genes was validated by quantitative real-time PCR. We found that there were a total of 7192 genes (5720 upregulated and 1472 downregulated, fold-change ≥ 2 or ≤ 0.5 for upregulation or downregulation, q-value < 0.05) differentially expressed in NaB-treated cells as compared with the untreated controls. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the differentially expressed genes were enriched in DNA replication, cell cycle, homologous recombination, pyrimidine metabolism, mismatch repair, and other signaling pathways and may take part in NaB-induced cell death. Among the identified factors, the MCM2-7 complex might be a target of NaB. Our findings provide an important basis for further studies of the complicate network that might regulate sensitivity of colorectal cancer cells to NaB.