Recently, multidrug-resistant clinical isolates of Acinetobacter baumannii have been found to have a high capacity to form biofilm. It is well known that bacterial cells within biofilms are highly resistant to antibiotics, UV light, acid exposure, dehydration, and phagocytosis in comparison to their planktonic counterparts, which suggests that the cells in a biofilm have altered metabolic activity. To determine which proteins are up-regulated in A. baumannii biofilm cells, we performed a proteomic analysis. A clinical isolate of A. baumannii 1656-2, which was characterized to have a high biofilm forming ability, was cultivated under biofilm and planktonic conditions. Outer membrane enriched A. baumannii 1656-2 proteins were separated by two-dimensional (2-D) gel electrophoresis and the differentially expressed proteins were identified by MALDI-TOF mass spectrometry. The proteins up-regulated or expressed only in biofilm cells of A. baumannii are categorized as follows: (i) proteins processing environmental information such as the outer membrane receptor protein involved in mostly Fe transport, a sensor histidine kinase/response regulator, and diguanylate cyclase (PAS-GGEDF-EAL domain); (ii) proteins involved in metabolism such as NAD-linked malate dehydrogenase, nucleoside-diphosphate sugar epimerase, putative GalE, ProFAR isomerase, and N-acetylmuramoyl-L: -alanine amidase; (iii) bacterial antibiotic resistance related proteins; and (iv) proteins related to gene repair such as exodeoxyribonuclease III and GidA. This proteomic analysis provides a fundamental platform for further studies to reveal the role of biofilm in the persistence and tolerance of A. baumannii.