The histones, that wrap the DNA to form the nucleosome core, are targeted by diverse post-translational modifications. How those modifications affect DNA accessibility and chromatin folding is a fundamental biological issue. Furthermore, tremendous amounts of data suggest that the loss of specific modifications as well as an inappropriate ectopic pattern of modifications at given loci contributes to the development of several human syndromes as well as cancers. In this review, we focus on the current knowledge of the regulation and impact of histone H4 lysine 16 acetylation on the genome. Recent discoveries highlight its crucial involvement in events such as transcription regulation, chromatin specialization, chromosome compaction and tumoral progression.