Bio-butanol is a potential energy which is similar to gasoline in properties such as energy density, and it can be substituted to petrol and diesel engine without any transformation, what is more, nothing that pollute the environment is emitted, so bio-butanol is considered as one of the most promising fuel in the coming future.At present, butanol production strains mainly include Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum and Clostridium saccharoperbutylacetonicum, and in this research, we identified another butanol production strain Paenibacillus Polymyxa.Different soil samples have been collected from six places, after several times enrichment culture, and purify for several times till the colony and bacteria are observed the same, finally, 17 strains have been isolated.Each of the strain is inoculated to liquid fermentation medium, after culturing for 96h, samples are taken from the broth, and dectected with gas chromatograph (GC) to see the butanol concentration each of them.The result showed that one strain number as 10 possess the highest butanol prodution, which reach to 4.208g/L, very much higher than the second one whose yield is 2.584g/L, so the number 10 strain is regard as the target strain, and named as HIT1.Observation of morphology and analysis of physiology and biochemistry suggest HIT1 to be facultative anaerobic, Gram negative, flagellate, which is able to use glucose, lactose, maltose, salicin, mannose, sorbierite and starch, while unable to use ribose, casein, lipase and indol.HIT1 is also able to hydrolyze and liquefaction gelatine.Compare with the identification feature list on Bergeys Manual of Determinative Bacteriology, and the analysis of 16SrDNA sequence showed that HIT1 belongs to Paenibacillus Polymyxa.With the optimizing assay, the optimum concentration of glucose is 25g/L, while the initial pH is 6.5,and HIT1 utilize tryptone better than yeast extract.