Abstract: Objective To determine the genes with high sensitivity and specificity in colorectal cancer based on bioinformatics and molecular biological experiments. Methods Bioinformatics software was used to analyze the highly expressed biomarker aspartate beta-hydroxylase domain-containing protein 1 (ASPHD1) in colorectal cancer tissues. The expression of ASPHD1 in colorectal cancer cell lines and its effects on cell viability were measured by real-time fluorescence quantitative PCR and CCK-8 assay, respectively. Results The levels of ASPHD1 in 275 samples of colon adenocarcinoma, 349 samples of normal colon, 92 samples of rectal adenocarcinoma and 318 samples of normal rectal tissue were investigate using GEPIA database. Compared with normal tissues, the expression of ASPHD1 in colorectal cancer tissues significantly increased. The overall survival rate of colon adenocarcinoma patients with high expression of ASPHD1 was significantly lower than those with low expression of ASPHD1 (P<0.05). ASPHD1 was associated with clinical stage, and lymph node and distant metastasis (P<0.05). ASPHD1 was positively correlated with the expression of Vimentin encoding gene Vim, a marker of epithelial-mesenchymal transition (R=0.11, P<0.05). The results of real-time fluorescence quantitative PCR showed that the levels of ASPHD1 in intestinal cancer cell lines SW480, SW620, HCT116 and LoVo increased compared with those in human normal intestinal epithelial cells NCM460. Small interfering RNA of ASPHD1 significantly inhibited the cell viability of HCT116 and LoVo. Conclusions ASPHD1 is highly expressed in colorectal cancer tissues, which is related to the prognosis of colorectal cancer and affects the cell viability of intestinal cancer cells. It can be used as a biomarker for colorectal cancer and provide new thoughts for clinical treatment.