Significance The small GTPase Ran plays fundamental roles in cellular processes such as nucleo-cytoplasmic transport, mitotic spindle formation, and nuclear envelope assembly. Recently, Ran was found to be lysine acetylated, among others, in functionally important regions such as switch I and switch II. Using the genetic code expansion concept we show that lysine acetylation affects many important aspects of Ran function such as RCC1-catalyzed nucleotide exchange, intrinsic nucleotide hydrolysis, import/export complex formation, and Ran subcellular localization. Finally, we present evidence for a regulation of Ran acetylation by sirtuin deacetylases and lysine acetyltransferases.