Trypanosoma cruzi is the causal agent of Chagas’ disease, which is one of the biggest public health problems in Latin America. Several post-translational modifications (PTM) have been studied in T. cruzi but there is no work focused on the O-GlcNAcylation, which is a highly conserved monosaccharide PTM found on serine and threonine residues of proteins mainly from the nucleus, cytoplasm and mitochondria. It is thought to regulate proteins in a manner analogous to protein phosphorylation, even their crosstalk allows the regulation of cellular functions in response to nutrients and stress. In the present work we demonstrate O-GlcNacylation in T. cruzi epimastigotes using two different methods: western blot with specific antibodies and click chemistry labelling. Then, we identify 2033 O-GlcNAcilated proteins and 48 modification sequences by mass spectrometry. Most of this proteins are part of structures, functions and pathways that are essentials for the parasite survival and evolution. For the first time O-GlcNacylation is idenytified in T. cruzi proteins and due to the many biological processes in which they participate, this study opens a new research field in Trypanosomatids biology, infection processes and potential identification of therapeutic targets.