A novel Gram-positive, fructophilic, catalase negative, and rod-shaped strain, designated strain 10HT was isolated from kôso, a Japanese sugar-vegetable fermented beverage obtained from a food processing factory in Saku City, Nagano Prefecture, Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed strain 10HT to belong to the genus Lactobacillus, with closely related type strains being Lactobacillus kunkeei YH-15T (95.5% sequence similarity), Lactobacillus ozensis Mizu2-1T (95.4% sequence similarity), and Lactobacillus apinorum Fhon13NT (95.3% sequence similarity). The isolate was found to grow at 18-39 °C (optimum 27 °C), pH 4.0-7.0 (optimum pH 6.5) and in the presence of 0-2% NaCl (optimum 0% NaCl). The G + C content of its genomic DNA was determined to be 30.5 mol%. The major fatty acid (≥ 10%) components identified included C16:0, C19:0 cyclo ω7c, C19:0 cyclo ω9c, and C18:1 ω9c. The polar lipids were identified as lysophosphatidylethanolamine, phosphatidylethanolamine and glycolipids. The predominant isoprenoid quinones (> 10%) were identified as MK-7, MK-8, MK-9 and MK-10. The amino acid composition of the cell wall was detected as comprising Asp, Glu, Ala, and Lys but the strain lacks meso-diaminopimelic acid. As with other fructophilic lactic acid bacteria, such as L. kunkeei and L. apinorum, strain 10HT was found to prefer D-fructose to D-glucose as a growth substrate. On the basis of these genetic and phenotypic results, the isolate is concluded to represent a novel species, for which the name Lactobacillus kosoi is proposed. The type strain is 10HT (= NBRC 113063T = BCRC 81100T).