Gli1 is a transcription factor and oncogene with documented roles in the progression of several cancer types, including cancers of the skin and pancreas. The contribution of Gli1 to the progression of breast cancer is less established. In order to investigate the functional impact of Gli1 in breast cancer, expression of Gli1 and its contribution to cell growth was assessed in breast cancer cell lines. These in vitro results were compared to expression of Gli1, determined by immunohistochemistry, in 171 breast cancers. In these cancers, the association of Gli1 with expression of estrogen receptor α (ERα) and progesterone receptor (PR), ErbB2, p53, the rate of proliferation, and clinicopathologic parameters and outcome was assessed. Expression of Gli1 and ERα mRNA was strongly correlated in ERα-positive cell lines (r = 0.999). Treatment with estrogen increased expression of Gli1 in 2 of 3 ERα-positive cell lines; this increase was prevented by treatment with the ERα-specific antagonist MPP. Silencing of Gli1 by shRNA markedly reduced the survival of two ERα-negative cell lines, but caused only a modest reduction in ERα-positive cell lines. In breast cancer tissues, cancers with nuclear localization of Gli1 had a higher ERα (P=0.027) and lower p53 expression (P=0.017) than those without nuclear localization of Gli1. However, nuclear localization of Gli1 was predictive of a poorer cancer-specific survival in ERα-negative, including triple negative, cancers (P = 0.005), but not ERα-positive cancers. In conclusion, we demonstrate a positive association between expression of Gli1 and ERα; however, our data indicate a greater functional effect of Gli1 in ERα-negative cancers. © 2009 Springer Science+Business Media, LLC.