Hepatitis B virus (HBV) X protein (HBx) is a 17-kDa transcriptional coactivator that plays a significant role in the regulation of genes involved in inflammation and cell survival. It has been known to be involved in the development of liver cancer and alteration of the cellular HBx level may influence the pathogenesis of HBV-induced liver diseases. The transcription factor GLI1, a member of the glioma-associated oncogene homologue (GLI) subfamily of Krüppel-like zinc finger proteins is involved in signal transduction within the hedgehog (Hh) signaling pathway, which is involved in the development of many human malignancies. GLI activation is important for cell proliferation and anti-apoptosis in various cancers. To investigate whether the transcriptional coactivator HBx binds to the zinc finger transcription factor GLI1, recombinant HBx and GLI1 were isolated. Expression and purification of the HBx and GLI1 proteins were successfully performed in Escherichia coli. The binding of HBx to GLI1 was detected by surface plasmon resonance spectroscopy (BIAcore), fluorescence measurement, and a His-tagged pull-down experiment. After measuring the fluorescence emission spectra of purified HBx and GLI1, it was found that the interaction of these proteins is accompanied by significant conformational changes in one or both. This study provides important clues for the structural identification of signal transduction pathways involving the HBx and GLI1 proteins.