Acetylation is a post-translational modification that influences the activity of numerous proteins in vitro. Among them, the myogenic transcription factor MyoD displays an increased transcriptional activity in vitro when acetylated on two lysines, lysine 99 and 102. Here, we have investigated the biological relevance of this acetylation in vivo. Using specific antibodies, we demonstrate that endogenous MyoD is acetylated on lysine 99 and 102 in myoblasts. Moreover, we show the functional importance of acetylation in live animals, using a mutant of MyoD in which lysines 99 and 102 were replaced by arginines. Knock-in (KI) embryos homozygous for the MyoD R 99,102 allele expressed slightly reduced levels of MyoD, but they developed normally. However, the KI homozygous adult mice showed a phenotype close to that of MyoD knock-out animals, including delayed muscle regeneration in vivo, and increased numbers of myoblasts but with reduced differentiation potential in vitro. Taken together, these results demonstrate the importance of MyoD acetylation for adult myogenesis