Aberrant activation of the Wnt/β-catenin signaling pathway plays a crucial role in the development and progression of colorectal cancer. Previously, we identified a set of candidate genes that were regulated by this signaling pathway, and we focused on MOSPD1, motile sperm domain containing 1, in this study. Immunohistochemical staining revealed that the expression of MOSPD1 was elevated in tumorous cells of colorectal cancer tissues compared with non-tumorous cells. Using ChIP-seq data and JASPAR database, we searched for the regulatory region(s) in the MOSPD1 gene as a target of the Wnt/β-catenin signaling, and identified a region containing three putative TCF-binding motifs in the 3’-flanking region. Additional analyses using reporter assay and ChIP-qPCR suggested that this region harbors an enhancer activity through an interaction with TCF7L2 and β-catenin. These data have clarified that MOSPD1 is a novel direct target of the Wnt/β-catenin signaling. In addition, the identification of its enhancer region may be helpful for the future studies of precise regulatory mechanisms of MOSPD1.