DNA methylation regulates chromatin structure and function, playing key roles during development and in diseases. Since Tahiliani et al. identified TET1 as the first hydroxylase of 5mC in 2009, DNA hydroxymethylation, an important regulating way for DNA methylation, has become the hotspot of epigenetics. Here, the recent research progress of DNA hydroxymethylation in mouse embryonic stem cells was summarized.