The molecular interplay between nucleosomal packaging and the chromatin landscape regulates the transcriptional programming and biological outcomes of downstream genes. An array of epigenetic modifications plays a pivotal role in shaping the chromatin architecture, which controls DNA access to the transcriptional machinery. Acetylation of the amino acid lysine is a widespread epigenetic modification that serves as a marker for gene activation, which intertwines the maintenance of cellular homeostasis and the regulation of signaling during stress. The biochemical horizon of acetylation ranges from orchestrating the stability and cellular localization of proteins that engage in the cell cycle to DNA repair and metabolism. Furthermore, lysine acetyltransferases (KATs) modulate the functions of transcription factors that govern cellular response to microbial infections, genotoxic stress, and inflammation. Due to their central role in many biological processes, mutations in KATs cause developmental and intellectual challenges and metabolic disorders. Despite the availability of tools for detecting acetylation, the mechanistic knowledge of acetylation-mediated cellular processes remains limited. This review aims to integrate molecular and structural bases of KAT functions, which would help design highly selective tools for understanding the biology of KATs toward developing new disease treatments.