Trypanosomatids, which include major pathogens of humans and livestock, are flagellated protozoa for which cell cycle controls and the underlying mechanisms are not completely understood. Here, we describe a genome-wide RNA-interference library screen for cell cycle defects in Trypanosoma brucei. We induced massive parallel knockdown, sorted the perturbed population using high-throughput flow cytometry, deep-sequenced RNAi-targets from each stage and digitally reconstructed cell cycle profiles at a genomic scale; also enabling data visualisation using an online tool ( https://tryp-cycle.pages.dev/ ). Analysis of several hundred genes that impact cell cycle progression reveals >100 flagellar component knockdowns linked to genome endoreduplication, evidence for metabolic control of the G1-S transition, surface antigen regulatory mRNA-binding protein knockdowns linked to G2M accumulation, and a putative nucleoredoxin required for both mitochondrial genome segregation and for mitosis. The outputs provide comprehensive functional genomic evidence for the known and novel machineries, pathways and regulators that coordinate trypanosome cell cycle progression.