Proteomic analysis was applied to investigate the mechanism of the stimulatory effect of Tween 80 on the mycelial growth and exopolysaccharide production by an edible mushroom Pleurotus tuber-regium. 32 differentially expressed proteins were identified by one-dimension gel electrophoresis. Combined with our previous findings, the up-regulation of heat shock proteins might help to maintain cellular viability under environmental stress. The up-regulation of ATP:citrate lyase isoform 2 could suppress the activity of tricarboxylic acid cycle and, consequently, stimulate exopolysaccharide production. The present results provide important insight to the mechanism by which stimulatory agents (Tween 80) can increase the production of useful fungal metabolites and also fill the gap of our knowledge on the under-developed mushroom proteomics.