Acinetobacter baumannii is considered a problematic Gram-negative pathogen due to its widespread resistance to antibiotics. Understanding of resistance mechanisms in A. baumannii is critical for designing new and effective therapeutic options. However, this is hampered by the lack of tools to carry out genetic manipulations in A. baumannii. Here, we describe methods to use a chromosomal mini-Tn7-based single-copy gene expression system in A. baumannii. This system can be effectively used for performing genetic complementation studies, for tagging with fluorescent proteins, or for reporter fusion assays.