Transcription-factor-induced reprogramming of somatic cells to pluripotency is a very inefficient process, probably due to the existence of important epigenetic barriers that are imposed during differentiation and that contribute to preserving cell identity. In an effort to decipher the molecular nature of these barriers, we followed a genome-wide approach, in which we identified macrohistone variants (macroH2A) as highly expressed in human somatic cells but downregulated after reprogramming to pluripotency, as well as strongly induced during differentiation. Knockdown of macrohistone variants in human keratinocytes increased the efficiency of reprogramming to pluripotency, whereas overexpression had opposite effects. Genome-wide occupancy profiles show that in human keratinocytes, macroH2A.1 preferentially occupies genes that are expressed at low levels and are marked with H3K27me3, including pluripotency-related genes and bivalent developmental regulators. The presence of macroH2A.1 at these genes prevents the regain of H3K4me2 during reprogramming, imposing an additional layer of repression that preserves cell identity. Display omitted ► Macrohistone variants are regulated during reprogramming and differentiation ► The levels of macrohistone variants modulate the efficiency of reprogramming ► MacroH2A.1 occupies pluripotency and differentiation genes in keratinocytes ► MacroH2A.1 occupancy prevents the gain of H3K4me2 during reprogramming The epigenetic mechanisms responsible for maintaining the identity of somatic cells are not fully understood. Here, Izpisua Belmonte and colleagues show that macrohistone variants play an important role in preserving somatic cell identity. Macrohistone variants occupy the regulatory regions of bivalent and pluripotency-related genes in somatic cells, where they prevent the regain of the active chromatin mark H3K4me2 during the process of reprogramming to pluripotency.