The optimal temperature for the microbial polysaccharide fermentation is no higher than 30 °C, which is economically undesirable due to additional cooling cost. To solve this problem in the case of welan gum production, we obtained the high-temperature-tolerant-producing strain, Sphingomonas sp. HT-1 by atmospheric and room-temperature plasma-induced mutation. Using HT-1, we obtained a concentration and 1 % aqueous viscosity of 26.8 ± 0.34 g/L and 3.50 ± 0.05 Pa s at a comparatively higher optimal temperature (37 °C). HT-1 was further characterized to understand the mechanism by which these properties are improved. Results indicated that high yield could be attributed to the following: (1) enhanced intracellular synthesis, demonstrated by an increase in the activities of key enzymes, and (2) accelerated cross-membrane substrate uptake and product secretion, indicated by improved membrane fluidity and permeability. Temperature tolerance could be attributed to the overexpression of the investigated heat shock proteins and oxidative stress proteins.