2-Deoxy-D-glucose (2-DG) is a non-metabolizable glucose analogue and competitive inhibitor of glycolysis. Effect of 2-DG on gellan gum biosynthesis by Sphingomonas paucimobilis ATCC31461 were studied in this research. The concentration and the addition time of 2-DG significantly affected the biomass and gellan gum accumulation. The maximum gellan gum yield of 20.78 g/L was obtained with the addition of 50 µg/L of 2-DG at 24 h. The mechanism of 2-DG addition favoring to gellan production was revealed by determining the activities of key enzymes. Results indicated that 2-DG addition increased the activities of glucosyltransferase and inhibited UDP-glucose pyrophosphorylase activity. The result indicated that 2-DG inhibited glycolysis and changed metabolic driving force to activate gellan gum biosynthesis metabolism pathways.