Due to the complex mechanisms involved in butanol-induced stress response, butanol tolerance phenotype is difficult to engineer even in microorganisms with well-defined genetic backgrounds. We therefore aimed to isolate butanol-tolerant microorganisms from environmental samples as potential alternative hosts for butanol production. Soil samples collected were subjected to butanol stress. A microbial strain capable of 2.5-3 % (w/v) butanol tolerance was isolated and identified as Enterococcus faecium by 16S rDNA analysis. The isolate grew readily under both aerobic and anaerobic conditions and was capable of producing butanol anaerobically. In comparison with the obligate anaerobe Clostridium acetobutylicum, the growth under both aerobic and anaerobic conditions of the isolated strain, together with no detection of butyrate and lack of two-phase fermentation suggests different metabolic networks from the obligate anaerobe C. acetobutylicum. Under anaerobic condition, butanol reached up to 0.4 g l(-1) in a batch culture without heterologous introduction of butanol biosynthetic pathway. Besides butanol tolerance, the isolated E. faecium IB1 showed high tolerance to 10 % (w/v) ethanol and 3 % (w/v) isobutanol. With distinct features including high butanol tolerance and natural butanol production, the isolated E. faecium IB1 with minimum metabolic engineering can be explored as a potential host for butanol production.