Genetic studies have revealed essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in Caenorhabditis elegans. However, large-scale identification of O-GlcNAcylated proteins and mapping the modification sites in C. elegans remain relatively unexplored. By using a chemoproteomic strategy, we herein report the identification of 108 high-confidence O-GlcNAcylated proteins and 64 modification sites in C. elegans. Furthermore, quantitative proteomics upon altering O-GlcNAcylation show that the abundance of a large number of proteins are affected by O-GlcNAc. These proteins are involved in regulating reproduction and lifespan, which may correlate with the previously observed phenotypes in genetic studies. The data set in this study reveals the O-GlcNAc modification landscape in C. elegans and provides a valuable resource for dissecting the biological function of O-GlcNAcylation.