The thermal stability of polysaccharides under heat treatment is an important factor to their functionality in food and pharmaceutical fields. The stiff branched beta-glucan coded as AF1-1 isolated from Auricularia auricula-judae was investigated with viscometry, dynamic light scattering (DLS), and size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS) in water at 25 to 170 degrees C. The chain conformation of AF1-1 in the aqueous solution exhibited a sharp decrease in viscosity, hydrodynamic radius (R-h), and weight-average molecular weight (M-w) at elevated temperature in a narrow range of 140 to 160 degrees C. It was confirmed that the conformation transitions of the AF1-1 chains from rod-like chains to the flexible occurred during heating to 140-160 degrees C for 30 min, leading to the coexistence of the flexible chains and stiff chains at 155 degrees C as a result of the breaking of the intra- and intermolecular hydrogen bonds of the AF1-1 macromolecules. The results from scanning electron microscopy and atomic force microscopy further directly proved that the AF1-1 nanofibers in water were destructed into flexible coils consisting of individual chain at the elevated temperature higher than 155 degrees C, supporting the conformation transition. The conformational transition from stiff to flexible chains at 140-160 degrees C was irreversible. However, the chain shape and stiffness of AF1-1 was stable below 140 degrees C and hardly changed with an increase in the temperature. This was important for the application in the fields of food and pharmaceutical.