In the present study, the cultural conditions for exopolysaccharide (EPS) production from probiotic yeast Lipomyces starkeyi VIT-MN03 were optimized using response surface methodology (RSM) to maximize the yield of EPS. Interactions among the various factors viz. sucrose concentration (1–3 g{\%}), NaCl concentration (2–4 g{\%}), pH (3–5), temperature (20–30 °C) and incubation period (20–40 days) during EPS production were studied using Box-Behnken design (BBD). The EPS was purified and characterized using various instrumental analyses. The properties like adhesion, antioxidant, biosurfactant, cholesterol removal and binding ability to mutagens were also tested for EPS produced. Sixfold increase in EPS production (4.87 g L −1 ) by L. starkeyi VIT-MN03 was noted under optimized condition. EPS showed a high viscosity (1.8 Pa S −1 ) and good shear-thinning properties. Instrumental analysis showed that EPS was heteropolysaccharide composed of glucan, mannan and rhamnan. Lipomyces starkeyi VIT-MN03 exhibited good self-adhesion (95{\%}) and co-aggregation ability (93{\%}). Adhesion efficiency for yeast inoculum containing 5.5 × 10 7 CFU mL −1 per 9.2 cm 2 of Caco-2 cell (colorectal adenocarcinoma) was noted. The probiotic EPS displayed strong antioxidant ability to scavenge hydroxyl radical and DPPH by 58{\%} and 71{\%} respectively. In addition, biosurfactant activity (86{\%}) and cholesterol removal (90{\%}) ability of probiotic EPS was also tested. EPS bound cells of L. starkeyi VIT-MN03 showed good binding ability to mutagens. These results support the effectiveness of using RSM for maximum EPS production. To the best of our knowledge, this is the first report on optimization of EPS production by probiotic yeast.