Psychrophilicity of Gram-positive bacterium, Bacillus psychrosaccharolyticus was investigated in a proteomic approach. One hundred and thirty-one protein spots were analyzed by electrospray ionization-quadrupole-time of flight-tandem mass spectrometry and identified using an unpublished translated contig database as well as a nonredundant Gram-positive bacteria protein database from NCBI because of the lack of a genome sequence of this organism. Results focused on proteomic behavior of cold-response show that global up-regulation of metabolic functions and protective mechanism by stress responses might play a major role in psychrophilicity of B. psychrosaccharolyticus.