DIXDC1, a novel type of DIX domain-containing protein in zebrafish, is the human homolog of Ccd1. It upregulates the Wnt signaling pathway functioning downstream of Wnt and upstream of axin. Here we aimed to efficiently generate a monoclonal antibody against DIXDC1 protein, using it as a tool to study the role of DIXDC1 in colonic tumorigenesis. We transformed the plasmids of PGEX-DIXDC1-C and PGEX-DIXDC1-N separately into Escherichia coli BL21 (DE3) for expression, and then injected the purified GST-DIXDC1 fusion protein into Balb/c mice. Hybridomas were screened by indirect enzyme-linked immunosorbent assay with purified fusion protein. Eight hybridoma clones effective in detecting the DIXDC1 protein were characterized by immunohistochemistry and Western immunoblotting. By using the MAbs, we found that the DIXDC1 protein was overexpressed in human colorectal cancer tissues. Taken together, the MAbs would be helpful for understanding the distribution and functions of DIXDC1.