The parasite Trypanosoma brucei periodically changes the expression of its protective variant surface glycoproteins (VSGs) to evade the host’s immune system in a process known as antigenic variation. One route to change VSG expression is by transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machinery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We identified several novel DOT1B interactors. One of these was the trimeric Ribonuclease H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage and recombination-based ES switching events. Surprisingly, the observed VSG deregulation was similar to Ribonuclease H2 mutants. We here propose that the epigenetic regulator DOT1B forms a complex with RNase H2 that acts in resolving R-loops to ensure genome integrity and contribute to the tightly regulated process of antigenic variation.