Abstarct: In this study, exopolysaccharide (EPS) was produced by Rhodotorula minuta in a medium contained (g/l) yeast extract, 18.75; xylose, 6.25; (NH4)2SO4, 2.5; KH2PO4, 1; MgSO4.7H2O, 0.5; NaCl, 0.1 and CaCl2.2H2O, 0.1. The yield of crude and purified EPS were obtained 3/511 g/l and 2/081 g/l respectively. The structural characterization was investigated by methylation analysis. The results revealed that backbone of the pure EPS is mannose unit with (1→3) and (1→4) lixnkage, which in some of the main chain points at C-6 of mannose there are side branches with repeating unit of glucose. At the first step, various carbon (starch, glucose, fructose, lactose, sorbitol) and nitrogen sources (peptone casein, peptone beef, NH4NO3, soybean, urea, yeast extract) were investigated to evaluated the polysaccharide content. One factor at time method was applied to select key substrates and subsequently Response Surface Methodology (RSM) was used to optimize the level of key substrates. By using the one factor at time method, starch (carbon source) and soybean (nitrogen source) were chosen as good substrates for EPS production. The maximum EPS yield (13/4 g/l) was obtained that had 15 g/L starch and 30 g/L soybean via Response Surface Methodology.