Xanthan is one of the most important biopolymers characterised by a high commercial value. Since the availability of essential nutrients influences its biosynthesis, the aim of this study was the optimisation of carbon, organic and inorganic nitrogen content in glycerol-based medium employing response surface methodology. The cultivation of strain Xanthomonas campestris ATCC 13951 was carried out under appropriate conditions on media with glycerol, peptone and ammonium-nitrate formulated according to Box-Behnken design (33), while the desirability function approach was used for determination of optimal nutrient levels. The final model predicts that the maximal amount of xanthan (12.95 g/L) is produced when the initial contents of glycerol, peptone and ammonium-nitrate in the medium are 32.96 g/L, 0.55 g/L and 0.73 g/L, respectively. To minimize the residual nutrient content and therefore the costs of effluent processing, additional optimisation was performed. In order to validate the optimisation model developed and examine the bioprocess success with crude glycerol as the sole carbon source, additional experiments were performed. The results represent reliable information for further investigations.