Inducible nitric oxide synthase (iNOS) plays a central role in tissue damage during endotoxic shock. However, the underlying mechanisms that control transcription of iNOS are not completely defined. A screening strategy with DNA pull-down technology and two-dimensional difference in gel electrophorcsis (2D-DIGE) analysis was used to identify regulators that interact with the iNOS promoter. We found 14 proteins that bind to the iNOS promoter in the liver of endotoxic shock mice. After matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis, one of these proteins was identified as testis-specific high-mobility-group protein (tsHMG), an alternative splicing isoform encoded by the mitochondrial transcription factor A gene. We identified the binding sites of tsHMG on the iNOS promoter using a LiquiChip system, and further confirmed interactions between tsHMG and iNOS by RT-PCR, western blotting and immunofluorescence. Functional analysis by over-expression and RNA interference of tsHMG revealed that tsHMG regulates lipopolysaccharide-stimulated iNOS expression. These results indicate that tsHMG participates in modulation of iNOS expression in the early stage of endotoxic shock.