Clostridium beijerinckii is a promising industrial microorganism for its ability to produce butanol, acetone, and isopropanol using a wide range of substrates, including pentoses, hexoses, and starch, via fermentation. The ubiquitous and highly abundant glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is essential for most organisms’ energy and carbon metabolism, which plays a critical role in some industrial bacteria. It catalyzes the simultaneous oxidation and phosphorylation of D-glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate in the presence of inorganic phosphate and nicotinamide adenine dinucleotide (NAD⁺). We determined the crystal structure of the GAPDH from C. beijerinckii (C. beijerinckii GAPDH). C. beijerinckii GAPDH consists of an α-β-α domain which shares an evolutionarily conserved fold consisting of two juxtaposed domains, an N-terminal NAD+-binding domain (NBD) and a C-terminal catalytic domain (CD). These findings provide insight into the molecular mechanism of action and cofactor-binding of this important industrial bacterial enzyme.