Halobacterium salinarum is an extremely halophilic archaea, which is able to live in highly saline environments. In a recent study, several halophilic archaea were found to have the ability to biodegrade organic hydrocarbon pollutants, but protein information regarding hydrocarbon degradation and tolerance in halophilic archaea has been relatively rare. In this study, the protein expression profile of H. salinarum cultured under different diesel concentrations (0, 2 and 4%) was investigated by two-dimensional gel electrophoresis. Proteins which increased their expression levels in diesel media were identified using matrix-assisted laser desorption/ionization-time of flight and electrospray ionization-tandem mass spectrometry analysis. Among these, a protein spot (named D3UPCA) which was up-regulated about nine-fold and found to have COG3388, an uncharacterized protein conserved in archaea, was selected in order to further characterize its functions. The D3UPCA coding gene (named d3upca) was cloned and expressed in Escherichia coli, and purified by the glutathione-S-transferase-fusion method. The function of the protein was estimated using various bioinformatics tools and was predicted to be related to the regulation of transcription and/or translation of genes needed to tolerate stresses associated within the presence of diesel oil.