The life cycle of mammals begins when a sperm enters an egg. Immediately after fertilization, both the maternal and paternal genomes undergo dramatic reprogramming to prepare for the transition from germ cell to somatic cell transcription programs 1. One of the molecular events that takes place during this transition is the demethylation of the paternal genome 2,3. Despite extensive efforts, the factors responsible for paternal DNA demethylation have not been identified 4. To search for such factors, we developed a live cell imaging system that allows us to monitor the paternal DNA methylation state in zygotes. Through siRNA-mediated knockdown in zygotes, we identified Elp3/KAT9, a component of the elongator complex 5, to be important for paternal DNA demethylation. We demonstrate that knockdown of Elp3 impairs paternal DNA demethylation as indicated by reporter binding, immunostaining and bisulfite sequencing. Similar results were also obtained when other elongator components, Elp1 and Elp4, were knocked down. Importantly, injection of mRNA encoding the Elp3 radical SAM domain mutant, but not the HAT domain mutant, into MII oocytes before fertilization also impaired paternal DNA demethylation indicating that the SAM radical domain is involved in the demethylation process. Thus, our study not only establishes a critical role for the elongator in zygotic paternal genome demethylation, but also suggests that the demethylation process may be mediated through a reaction that requires an intact radical SAM domain.