During the pyruvate production by Torulopsis glabrata,the osmotic stress in culture broth gradually increased. As a result,cell growth and acid production slowed. The proteome of the T. glabrata under hyperosmotic stress was investigated by two-dimensional polyacrylamide gel electrophoresis( 2D-PAGE) and isobaric tag for relative and absolute quantitation( iTRAQ). Compared to normal condition( 860 mOsmol /kg),125,91,109 proteins were upregulated and 94,89,78 proteins were downregulated under osmolarity of 1765 mOsmol /kg,2603 mOsmol /kg,and 3324 mOsmol /kg. The protein expression involved in central metabolic pathways and energy metabolism showed apparently upregulation. Furthermore,the expression levels of superoxide dismutase and verprolin increased under hyperosmotic conditions,of which the former might due to the accumulation of reactive oxygen species( ROS),and the latter might be related to the accumulation of proline. The results presented here will facilitate the improvement of T.glabrata under hyperosmotic stress.