Acinetobacter baumanniiis a nosocomial pathogen highly resistant to environmental changes and antimicrobial treatments. Regulation of cellular motility and biofilm formation is important for its virulence, although it is poorly described at the molecular level. It has been previously reported thatAcinetobactergenus specifically produces a small positively charged metabolite, polyamine 1,3-diaminopropane, that has been associated with cell motility and virulence. Here we show thatA. baumanniiencodes novel acetyltransferase, Dpa, that acetylates 1,3-diaminopropane, directly affecting the bacterium motility. Expression ofdpaincreases in bacteria that form pellicle and adhere to eukaryotic cells as compared to planktonic bacterial cells, suggesting that cell motility is linked to the pool of non-modified 1,3-diaminopropane. Indeed, deletion ofdpahinders biofilm formation and increases twitching motion confirming the impact of balancing the levels of 1,3-diaminopropane on cell motility. The crystal structure of Dpa reveals topological and functional differences from other bacterial polyamine acetyltransferases, adopting a β-swapped quaternary arrangement similar to that of eukaryotic polyamine acetyltransferases with a central size exclusion channel that sieves through the cellular polyamine pool. The structure of catalytically impaired DpaY128Fin complex with the reaction product shows that binding and orientation of the polyamine substrates are conserved between different polyamine-acetyltransferases.