The present study aimed to identify differentially expressed genes (DEGs) in colorectal cancer (CRC) and provide novel prognostic biomarkers for CRC. The microarray dataset GSE41258 was used to screen DEGs of CRC. Subsequently, a protein‑protein interaction network of DEGs and Gene Ontology analysis were performed to identify hub genes and associated biological processes. Nebulette (NEBL) and complement C1q like 1 (C1QL1) were validated using reverse transcription‑quantitative polymerase chain reaction in patients with CRC. Survival analysis was performed for the two hub genes. GSE41258 dataset included 182 CRC samples and 54 normal tissues. A total of 759 DEGs, including 279 upregulated and 480 downregulated were screened between both groups. NEBL and C1QL1 were identified as the two hub genes and upregulated genes involved in various biological processes, including 'regulation of biological quality' and 'response to stimulus', respectively. Additionally, the overexpression of NEBL and C1QL1 in experimental validation was consistent with the aforementioned bioinformatics analysis results. Survival analysis suggested that overexpressed NEBL in patients with CRC was associated with a positive prognosis for overall survival. In conclusion, CRC was associated with a large group of DEGs. From the upregulated genes, overexpressed NEBL in patients CRC indicated a positive prognosis for overall survival and may be used as a prognostic biomarker for patients with CRC.