AbstractPalm kernel cake (PKC), a by-product of palm oil industry, contains glucose and mannose as hexose sugars. This study was performed to determine the feasibility of using PKC as a lignocellulosic substrate for biobutanol production by Clostridium saccharoperbutylacetonicum N1-4 in an acetone-butanol-ethanol (ABE) fermentation process. Moreover, the effect of tryptone-yeast extract-acetate (TYA) medium and P2 medium on biobutanol production was evaluated. Experimental results showed that butanol production of 3.05 g/L was obtained using mannose sugar, which was comparable to 3.61 g/L butanol production measured using glucose. Moreover, the maximum production of biobutanol (0.38 g/L) was obtained at a PKC concentration of 30%, indicating the possibility of PKC utilization in butanol production. ABE fermentation of PKC using distilled water, TYA medium, and P2 medium showed that the highest butanol production (0.26 g/L) with ABE production of 0.38 g/L was obtained when ABE fermentation was conducted in P2 medium.