A two-stage culture (with controlled sucrose concentrations and temperatures) of Sphingomonas paucimobilis for gellan gum production has been previously investigated. Herein, the mechanism of a two-stage culture favoring gellan gum overproduction was revealed by analysing the cell-membrane permeability and the proteomics for gellan gum biosynthesis. The two-stage culture, resulted in 79.8% increased content of unsaturated fatty acids, and 3.95% increased ratio of unsaturated to saturated fatty acids in the cell membrane. Moreover, cell membrane permeability increased and thus further enhanced gellan gum biosynthesis. Proteomic analysis results indicated that 13 identified protein spots were involved in energy generation, glycogen biosynthesis, and glycolysis. These findings revealed that two-stage culture impellel carbon flux flow toward gellan gum biosynthesis.