R‐spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β‐catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C‐mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C‐mannosylation sites at Trp153 and Trp156; however, it had been unclear whether these sites are C‐mannosylated or not. We demonstrated that Rspo3 was C‐mannosylated at both Trp153 and Trp156 by mass spectrometry. Using C‐mannosylation‐defective Rspo3 mutant‐overexpressing cell lines, we found that C‐mannosylation of Rspo3 promotes its secretion and activates Wnt/β‐catenin signaling.