Previous studies demonstrated that the GBX2 homeobox gene is consistently overexpressed in cultured human prostate cancer cell lines. In this study, the human GBX2 cDNA was cloned and a quantitative reverse transcription-PCR method used to demonstrate that GBX2 mRNA expression is enhanced in approximately 70% of human prostate cancer tissues compared with normal human prostate tissues. Purified recombinant GBX2 protein binds specifically to an ATTA motif within the promoter of the interleukin 6 (IL-6) gene. Using an antisense approach, down-regulation of the expression of GBX2 correlated with decreased expression of IL-6 and an inhibition of tumorigenicity of PC3 human prostate cancer cells. In addition, in vitro growth of the antisense clones was partially restored by exogenous addition of recombinant IL-6 protein to the culture media. These data demonstrated that enhanced GBX2 expression results in a stimulation of malignant growth of prostate cancer cells and that part of this stimulation involves up-regulation in the transcription of the IL-6 gene.