Given the growing concerns about dwindling global oil deposits and greenhouse gas emissions, nations are working to produce renewable liquid biofuels with physical and chemical characteristics similar to those of gasoline. One such biofuel is butanol. Butanol is less corrosive than other biofuels and miscible with gasoline and diesel, and its energy content is comparable to the content of gasoline. The solubility characteristics of butanol also allow it to be transported in existing fuel pipelines and tanks. One factor influencing the economic viability of butanol fermentation is the availability of affordable, sustainable substrates for butanol production at competitive prices. In this respect, inexpensive substrates such as lignocellulosic biomass may serve as viable alternatives to classical substrates such as corn, starch, sucrose, and glucose. Lignocellulosic biomass represents the most abundant renewable energy resource on the planet, and it can be obtained on a sustainable basis for the production of butanol. Yet, this material also presents challenges that must be addressed before it can be used for economical butanol production. The challenges include the heterogeneous sugar content of lignocellulosic biomass and the generation of microbial inhibitory compounds during the deconstruction of lignocellulosic biomass into monomeric sugars for microbial utilization. This chapter, therefore, details: (1) mixed-sugar fermentation by solventogenic clostridia; (2) the metabolic engineering of solventogenic clostridia to increase the butanol titer and butanol-to-acetone ratio; (3) the pretreatment production of microbial inhibitory compounds and their effects on cell growth and butanol fermentation; and (4) perspectives on the commercialization of fermentative butanol production and possible strategies for overcoming the challenges that currently prevent it.