The Wnt Inhibitory Factor 1 (Wif1), known to inhibit Wnt signaling pathways, is composed of a WIF domain and five EGF-like domains (EGF-LDs) involved in protein interactions. Despite the presence of a potential O-fucosylation site in its EGF-LDs III and V, the O-fucose sites occupancy has never been demonstrated for WIF1. In this study, a phylogenetic analysis on the distribution, conservation and evolution of Wif1 proteins was performed, as well as biochemical approaches focusing on O-fucosylation sites occupancy of recombinant mouse WIF1. In the monophyletic group of gnathostomes, we showed that the consensus sequence for O-fucose modification by Pofut1 is highly conserved in Wif1 EGF-LD III while it was more divergent in EGF-LD V. Using click chemistry and mass spectrometry, we demonstrated that mouse WIF1 was only modified with a non-extended O-fucose on its EGF-LD III. In addition, a decreased amount of mouse WIF1 in the secretome of CHO cells was observed when the O-fucosylation site in EGF-LD III was mutated. Based on sequence comparison and automated protein modeling, we suggest that the absence of O-fucose on EGF-LD V of WIF1 in mouse and probably in most gnathostomes, could be related to EGF-LD V inability to interact with POFUT1.