Leuconostoc mesenteroides is a heterofermentative Grampositive bacterium that plays key roles in fermentation of foods such as kimchi, sauerkraut, and milk, leading to the production of various organic acids and aromatic compounds. To study the microbiological and genomic characteristics of L. mesenteroides, we have developed a new chemically defined minimal medium by using the single omission technique. During the exponential cell growth, this species required glutamine, methionine, valine, and nicotinic acid as essential nutrients and 8 amino acids (arginine, cysteine, histidine, leucine, phenylalanine, proline, threonine, and tryptophan), 5 vitamins (ascorbic acid, folic acid, inosine, calcium panthothenate, and thiamine), and others (manganese, magnesium, adenine, uracil, and Tween 80) as supplemental nutrients. This medium is useful to study the metabolic characteristics of L. mesenteroides and to explain its role in food fermentation.