Ahmednagar district is largest district in Maharashtra occupying 17048 sq. km area. Shrirampur taluka is located in the northern part of Ahmednagar district covering 579.87 sq. km. Nitrogen fixation is an important process in plants providing nitrogen as the most valuable macronutrient required by the plants. Acetobacter diazotrophicus as an endophytic nitrogen fixing bacteria is collected from Shrirampur taluka of Ahmednagar district (M.S). Total 12 rhizospheric samples were collected from various localities of the taluka and soil testing was done to determine pH, Water holding capacity and moisture percentage. After isolation of bacteria from soil sample, cell morphology and colony morphology was studied. With the help of special media and morphological characters preliminary identification of A. diazotrophicus was done. Out of these 12 bacterial strains 4 and 7 did not show characters identical with A. diazotrophicus; while other strains resembled A. diazotrophicus. Cell size of A. diazotrophicus was ranging between 1.04 to 2.00 μm. Colonies were creamy white, cicular, small to large sized, opaque with smooth margins. A. diazotrophicus is able to increase nutrient supply, soil fertility and crop growth of sugarcane. The study of A. diazotrophicus will be useful for further researchers and it will be better alternative for chemical fertilizers.