Response surface methodology was used to determine the exopolysaccharide production of Lactobacillus brevis E25. The effects of three variables, temperature (30, 36 and 42 °C), incubation time (18, 33 and 48 h) and initial pH of growth medium (4.5, 5.5, and 6.5) were studied. Under optimum growth conditions, the amount of exopolysaccharide derived from Lactobacillus brevis E25 ranged from 10 to 35 gL-1. The size of EPS molecules ranged from 10 5 to 10 7 Da. Infra red spectrum analysis showed characteristics absorption peaks indicating the presence of-OH, C=O and C-H groups. Furthermore, only glucose was detected as monosaccharide in exopolysaccharide structure, revealing that the structure of exopolysaccharide is a homopolymeric glucan type. Based on the differential scanning calorimeter thermograms, exopolysaccharide's melting temperature was observed around 116 °C.