Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli that use the 6-phosphogluconate/phosphoketolase (6-PG/PK) pathway as their central metabolic pathway and are reportedly unable to grow on fructose as a sole carbon source. We isolated a variant PM1 strain capable of sporadic growth on fructose medium and observed its distinctive characteristics of fructose metabolism. The end product pattern was different from what is expected in typical group III lactobacilli using the 6-PG/PK pathway (i.e., more lactate, less acetate, and no mannitol). In addition, in silico analysis revealed the presence of genes encoding most of critical enzymes in the Embden-Meyerhof (EM) pathway. These observations indicated that fructose was metabolized via two pathways. Fructose metabolism in the PM1 strain was influenced by the activities of two enzymes, triosephosphate isomerase (TPI) and glucose 6-phosphate isomerase (PGI). A lack of TPI resulted in the intracellular accumulation of dihydroxyacetone phosphate (DHAP) in PM1, the toxicity of which caused early growth cessation during fructose fermentation. The activity of PGI was enhanced by the presence of glyceraldehyde 3-phosphate (GAP), which allowed additional fructose to enter into the 6-PG/PK pathway to avoid toxicity by DHAP. Exogenous TPI gene expression shifted fructose metabolism from heterolactic to homolactic fermentation, indicating that TPI enabled the PM1 strain to mainly use the EM pathway for fructose fermentation. These findings clearly demonstrate that the balance in the accumulation of GAP and DHAP determines the fate of fructose metabolism and the activity of TPI plays a critical role during fructose fermentation via the EM pathway in L. panis PM1. L actobacilli generate metabolic energy (i.e., ATP) mainly by substrate-level phosphorylation by relatively simple pathways: the Embden-Meyerhof (EM) pathway and/or the 6-phosphogluconate/phosphoketolase (6-PG/PK) pathway. The members of the genus Lactobacillus are subdivided into three groups according to their fermentative characteristics on the basis of the presence of these pathways (1, 2). Group I lactobacilli are obligatory homofermentative lactobacilli that exclusively ferment hexose sugars (e.g., glucose) to lactate via the EM pathway; however, they do not metabolize pentose sugars or gluconate. Group II lactobacilli are the facultative heterofermentative lactobacilli that ferment hexoses to lactate via the EM pathway. They can also ferment pentose sugars using an inducible phosphoketolase, producing lactate and acetate, and they can yield carbon dioxide from gluconate but not from glucose. Group III lactobacilli are the obligatory heterofermentative lactobacilli that ferment hexoses to lactate, ethanol and/or acetate, and carbon dioxide, which is a distinct feature of the group III lactobacilli. Group III lactobacilli exclusively use the 6-PG/PK pathway to achieve this metabolism.Fructose, an abundant hexose sugar found in many plants, is one of the main monosaccharides for bacteria...