Gluconacetobacter diazotrophiucus (Gd) is a N₂-fixing endophytic bacterium that colonizes intercellular spaces and vascular tissues from sugarcane, promoting its growth without inducing disease symptoms. This bacterium has been described as being able to form biofilms which is being considered a default way of life for bacteria and, like the endophytic, a fundamental one for biological association. Usually, this bacterium natural habitat is inhospitable for a free life and a biofim growth would act as a protection from hostile conditions. In this work biofilm formation was investigated using proteomic techniques by evaluating the proteins that were differentially expressed in the biofilm and planktonic systems. Gd (Pal 5) was grown in LGIP medium with 1mM ammonium sulfate until reaching the stationary phase. Biofilm growth was in the presence of glass wool and planktonic growth was in suspension. After being harvested by sonication, biofilm cells were washed with 100mM ammonium bicarbonate; cells in planktonic growth were harvested and washed as the biolfim cells. Proteins extracted from cells grown in both conditions were processed to a membrane free extract. 2D-gel electrophoresis was used to characterize each cell proteome. Proteins were identified after spots excision, trypsin digestion, MALDI-TOF-TOT PMF and MS/MS analysis and MASCOT search in NCBInr-data base. The gels had differential protein expression by Coomassie blue staining. Planktonics cells showed differential and increased protein expression related to nitrogen metabolism, especially nitrogen fixation whereas biofilm cells expressed proteins related to carbohydrate metabolism, e.g. glican biosynthesis and to folding and associated processes. Supported by CAPES, FAPERJ, CNPq.