Dot1/DOT1L catalyzes the methylation of histone H3 lysine 79 (H3K79), which regulates diverse cellular processes, such as development, reprogramming, differentiation, and proliferation. In regards to these processes, studies of Dot1/DOT1L-dependent H3K79 methylation have mainly focused on the transcriptional regulation of specific genes. Although the gene transcription mediated by Dot1/DOT1L during the cell cycle is not fully understood, H3K79 methylation plays a critical role in the progression of G₁ phase, S phase, mitosis, and meiosis. This modification may contribute to the chromatin structure that controls gene expression, replication initiation, DNA damage response, microtubule reorganization, chromosome segregation, and heterochromatin formation. Overall, Dot1/DOT1L is required to maintain genomic and chromosomal stability. This review summarizes the several functions of Dot1/DOT1L and highlights its role in cell cycle regulation.