Abstract Increasing environmental concerns regarding fossil fuels and potential future supply constraints have driven the exploration of alternative fuel resources. Using syngas to produce biofuels through microbial fermentation processes provides an excellent option for the synthesis of fuels and chemicals in a clean and sustainable way. The fermentation of syngas by anaerobic acetogens via the Wood-Ljungdahl pathway has attracted considerable interest to for the production of biofuels. The major natural fermentation products of these bacteria are acetate, butyrate, ethanol, butanol, and 2,3-butanediol, which can be used directly or serve as precursors for biofuel and industrial chemical production. However, the widespread use of acetogens as production biocatalysts has been partially limited by their metabolic and energetic constraints for efficient conversion of syngas into target products. A comprehensive understanding of the cellular biology that enables syngas fermentation by these versatile microorganisms is necessary to model the electron and carbon flow in specific production routes, which can contribute substantially to design strategies for acetogen cell engineering and to optimize these technologies to an industrially attractive production level. In this review, we summarize the metabolic and energy conservation mechanisms of most known acetogens during syngas fermentation and discuss parameters that can be modulated to improve their metabolic efficiencies. Finally, the potential to utilize metabolic engineering to improve the spectrum of acetogen products is discussed. This will be helpful in developing acetogens as efficient syngas fermentation biocatalysts for biofuel production in large-scale industrial processes and therefore act as a novel microbial production platform that is both environmentally safe and sustainable.