Indole-3-carbinol (I3C), a naturally occurring compound of Brassica vegetables, has promising anticancer properties and activates an anti-proliferative pathway that induces a G1 cell cycle arrest of human breast cancer cells. A microarray analysis of I3C treated versus untreated MCF-7 breast cancer cells revealed that I3C increased expression of the interferon gamma receptor 1 (IFNγR1). Western blot and RT–PCR analysis demonstrated that I3C strongly and rapidly stimulated IFNγR1 gene expression. Transfection of a series of 5′ deletion constructs of the IFNγR1 reporter plasmids revealed that I3C significantly stimulates the promoter activity of the IFNγR1 and uncovered an I3C-responsive region between −540 and −240 bp of the IFNγR1 promoter. I3C stimulation of the IFNγR1 expression suggests that indole treatment should enhance IFNγ responsiveness in breast cancer cells. A combination of I3C and IFNγ synergistically activated STAT1 proteins by increasing phosphorylation at the Tyr-701 site. In addition, I3C and IFNγ together more effectively induced a G1 cell cycle arrest and stimulated expression of the p21Waf1/Cip1 cell cycle inhibitor, compared with the effects of either agent alone. Our results suggest that one mechanism by which I3C mediates these anticancer effects is by stimulating expression of the IFNγR1 and augmenting the IFNγ response in MCF-7 human breast cancer cells.