Galectin-3 is expressed in a cell-type specific manner in human pituitary tumors and may have a role in pituitary tumor development. In this study, we hypothesized that Galectin-3 is regulated by RUNX proteins in pituitary tumors. Transcription factor prediction programs revealed several putative binding sites in the LGALS3 (Galectin-3 gene) promoter region. A human pituitary cell line HP75 was used as a model to study LGALS3 and RUNX interactions using Chromatin immunoprecipitation assay and electrophoresis mobility shift assay. Two binding sites for RUNX1 and one binding site for RUNX2 were identified in the LGALS3 promoter region. LGALS3 promoter was further cloned into a luciferase reporter, and the experiments showed that both RUNX1 and RUNX2 upregulated LGALS3. Knock-down of either RUNX1 or RUNX2 by siRNA resulted in a significant downregulation of Galectin-3 expression and decreased cell proliferation in the HP 75 cell line. Immunohistochemistry showed a close correlation between Galectin-3 expression and RUNX1/RUNX2 level in pituitary tumors. These results demonstrate a novel binding target for RUNX1 and RUNX2 proteins and suggest that Galectin-3 is regulated by RUNX1 and RUNX2 in human pituitary tumor cells by direct binding to the promoter region of LGALS3 and thus may contribute to pituitary tumor progression.