The superiority of biobutanol as a fuel for vehicles over other biofuels has been reported numerous times in recent years (Kumar and Gayen, 2011). Therefore, fermentative production of butanol has attracted the attention of various new research groups in academia and industries. In a review, we have already summarized the development and challenges in fermentative production of biobutanol until 2011 (Kumar and Gayen, 2011). In this chapter, we will highlight the further developments in this field along with the outstanding challenges. In spite of several advantages of butanol as fuel for vehicles, there are still some challenges in acetone–butanol–ethanol (ABE) fermentation in terms of low butanol titer, low productivity, and low yield, high cost of pretreatment of cellulosic feedstocks, presence of toxic compounds in feedstock hydrolysates, product inhibition, and costly and low efficiency recovery processes, which make biobutanol less competitive than other biofuels like bioethanol and biodiesel (Zheng et al., 2015). These challenges are being addressed using several strategies. Details of these strategies are discussed in different sections of this chapter.