Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a CoA-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing genes of adhE1 and ctfA-ctfB-adc from C. acetobutylicum ATCC 824, which enabled it to produce n-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n-butanol tolerance of C. cellulovorans, to fully utilize its n-butanol output potential. To further improve n-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as host for metabolic engineering. The n-butanol production from AECC of the engineered C. cellulovorans enhanced 138-fold from less than 0.025 g/L to 3.47 g/L. This method represents a milestone toward n-butanol production by CBP, using single recombinant clostridia. The engineered strain offers a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose.