Klebsiella pneumoniae was engineered to produce isobutanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and alpha-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, a dagger ldhA/pBR-iBO (ilvIH-ilvC-ilvD-kivd-adhA), produced isobutanol (160 mg l(-1)) from crude glycerol. To increase the yield of isobutanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating alpha-acetolactate decarboxylase (adc). This further engineering step improved the yield of isobutanol from 160 to 320 mg l(-1). This represents the first successful attempt to produce isobutanol from crude glycerol.