DOT1 is an evolutionarily conserved histone H3 lysine 79 (H3K79) methyltransferase. K79 methylation is associated with transcriptional activation, meiotic checkpoint control, and DNA double-strand break (DSB) responses. Trypanosoma brucei has two homologs, DOT1A and DOT1B, which are responsible for dimethylation and trimethylation of H3K76, respectively (K76 in T. brucei is synonymous to K79 in other organisms). K76 dimethylation is only detectable during mitosis, whereas trimethylation occurs throughout the cell cycle. Deletion of DOT1B resulted in dimethylation of K76 throughout the cell cycle and caused subtle defects in cell cycle regulation and impaired differentiation. RNAi-mediated depletion of DOT1A appears to disrupt a mitotic checkpoint, resulting in premature progression through mitosis without DNA replication, generating a high proportion of cells with a haploid DNA content, an unprecedented state for trypanosomes. We propose that DOT1A and DOT1B influence the trypanosome cell cycle by regulating the degree of H3K76 methylation.