Product inhibition by butanol and acetone is a known drawback in acetone-butanol-ethanol (ABE) fermentation. Extractive fermentation improves butanol production by several ABE-producing Clostridium spp., but only low volume ratios (<4) of extractant to broth (V-e/V-b) have been studied. Here, a novel extractive fermentation process was developed using Clostridium saccharoperbutylacetonicum N1-4 and a large V-e/V-b ratio. A mixture of oleyl alcohol-tributyrin (1:1 (v/v)) yielded high distribution coefficients for both butanol (3.14) and acetone (0.660). Although a fed batch culture using free cells and the oleyl alcohol-tributyrin mixture at a V-e/V-b ratio of 5 had a lag phase of >24 h, it produced a higher concentration of total butanol (i.e., butanol produced in all the phases per broth volume used) of 24.2 g/L-broth after 96 h compared with 14.4 g/L-broth at a V-e/V-b ratio of 1, resulting in a low butanol concentration in the aqueous phase. The use of cells immobilized with calcium alginate beads shortened the lag phase to <12 h. Butanol production was achieved not only in a 3-phase mode (extractant, beads, and tryptone-yeast extract-acetate (TYA) medium) but also in a 2-phase mode (extractant and beads containing TYA medium, without an aqueous phase) at a V-e/V-b ratio of 5, resulting butanol concentrations of 30.9 g/L-broth and 27.7 g/L-broth, respectively. The 3-phases fed-batch extractive fermentation at a V-e/V-b ratio of 10 showed a better performance compared with published reports: a total butanol concentration of 64.6 g/L-broth and a butanol yield to consumed sugar of 0.378 C-mol/C-mol. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.