Protein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets. Here, we report that the AGC essential kinase 1 (TcAEK1) exhibits a cytosolic localization and a higher level of expression in the replicative stages of the parasite. A CRISPR/Cas9 editing technique was used to generate ATP analog-sensitive TcAEK1 gatekeeper residue mutants that were selectively and acutely inhibited by bumped kinase inhibitors (BKIs). Analysis of a single allele deletion cell line (TcAEK1-SKO), and gatekeeper mutants upon treatment with inhibitor, showed that epimastigote forms exhibited a severe defect in cytokinesis. Moreover, we also demonstrated that TcAEK1 is essential for epimastigote proliferation, trypomastigote host cell invasion, and amastigote replication. We suggest that TcAEK1 is a pleiotropic player involved in cytokinesis regulation in T. cruzi and thus validate TcAEK1 as a drug target for further exploration. The gene editing strategy we applied to construct the ATP analog-sensitive enzyme could be appropriate for the study of other proteins of the T. cruzi kinome. IMPORTANCE Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.