Metacyclogenesis is one of the most important processes in the life cycle of Trypanosoma cruzi. In this stage, noninfective epimastigotes become infective metacyclic trypomastigotes. However, the transcriptomic changes that occur during this transformation remain uncertain. Illumina RNA-sequencing of epimastigotes and metacyclic trypomastigotes belonging to T. cruzi DTU I was undertaken. Sequencing reads were aligned and mapped against the reference genome, differentially expressed genes between the two life cycle stages were identified, and metabolic pathways were reconstructed. Gene expression differed significantly between epimastigotes and metacyclic trypomastigotes. The cellular pathways that were mostly downregulated during metacyclogenesis involved glucose energy metabolism (glycolysis, pyruvate metabolism, the Krebs cycle, and oxidative phosphorylation), amino acid metabolism, and DNA replication. By contrast, the processes where an increase in gene expression was observed included those related to autophagy (particularly Atg7 and Atg8 transcripts), corroborating its importance during metacyclogenesis, endocytosis, by an increase in the expression of the AP-2 complex subunit alpha, protein processing in the endoplasmic reticulum and meiosis. Study findings indicate that in T. cruzi metacyclic trypomastigotes, metabolic processes are decreased, and expression of genes involved in specific cell cycle processes is increased to facilitate transformation to this infective stage.