Abstract. C-mannosylation is a unique type of protein glycosylation with a mannose attached to the tryptophan residue via the C-C linkage. Our previous study revealed that dpy-19 like 3 (DPY19L3) acts as a C-mannosyltransferase in human cells. The present study hypothesized that RPE-spondin (RPESP) may be a substrate protein of DPY19L3-mediated C-mannosylation. RPESP has unknown biological functions and has two putative C-mannosylation sites at the W 80 and W 83 residues; however, to the best of our knowledge, C-mannosylation of RPESP has not previously been investigated. The present study suggested that RPESP is C-mannosylated at W 80 and W 83 in human cells, whereas gain-of-function experiments using S2 cells revealed that human DPY19L3 catalyzed the C-mannosylation of RPESP at W 83 but not W 80, which suggested substrate specificity. In addition, the present study detected mRNA expression levels of RPESP in various types of cancer cell lines and high expression levels of RPESP were revealed in certain colorectal cancer cell lines, suggesting that RPESP may have an association with the malignancy of colorectal cancers.