Aims: To determine optimal exopolysaccharide (EPS) production conditions of the mesophilic lactic acid bacterium strain Lactobacillus sakei 0-1 and to detect possible links between EPS yields and the activity of relevant enzymes. Methods and Results: Fermentation experiments at different temperatures using either glucose or lactose were carried out. EPS production took place during the exponential growth phase. Low temperatures, applying glucose as carbohydrate source, resulted in the best bacterial growth, the highest amounts of EPS and the highest specific EPS production. Activities of 10 important enzymes involved in the EPS biosynthesis and the energy formation of Lact. sakei 0-1 were measured. The obtained results revealed that there is a clear link for some enzymes with EPS biosynthesis. It was also demonstrated clearly that the presence of rhamnose in the EPS building blocks is due to high activities of the enzymes involved in the rhamnose synthetic branch. Conclusions: EPS production in Lact. sakei 0-1 is growth-associated and displays primary metabolite kinetics. Glucose as carbohydrate source and low temperatures enhance the EPS production. The enzymes involved in the biosynthesis of the activated sugar nucleotides play a major role in determining the monomeric composition of the synthesized EPS. Significance and Impact of the Study: The proposed results contribute to a better understanding of the physiological factors influencing EPS production and the key enzymes involved in EPS biosynthesis by Lact. sakei.