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 responses. Trypanosoma brucei has two homologues, DOT1A and DOT1B, which are responsible for di-methylation and tri-methylation of H3K76 respectively (K76 in T. brucei is synonymous to K79 in other organisms). K76 di-methylation is only detectable during mitosis whereas tri-methylation occurs throughout the cell cycle. Deletion of DOT1B resulted in di-methylation 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.