Cytochrome P450-1A1 (CYP1A1) is an extrahepatic phase I metabolizing enzyme whose expression is suppressed under physiologic conditions but can be induced by substrates via the aryl hydrocarbon receptor (AhR). Recent studies have shown that the majority of breast cancer tumors constitutively express CYP1A1. These findings led us to test the hypothesis that CYP1A1 promotes breast cancer progression by evaluating the effects of CYP1A1 knockdown on the proliferation and survival of the MCF7 and MDA-MB-231 lines. Independently of estrogen receptor status, CYP1A1 knockdown decreased colony formation, decreased cell proliferation, blocked the cell cycle at G0-G1 associated with reduction of cyclin D1, and increased apoptosis associated with reduction of survivin. CYP1A1 knockdown markedly increased phosphorylation of AMPactivated protein kinase (AMPK) and decreased phosphorylation of AKT, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and 70-κDa ribosomal protein S6 kinase (P70S6K). AMPK inhibition by compound C partially abrogated the proapoptotic effects of CYP1A1 knockdown, suggesting that effects of CYP1A1 knockdown are mediated in part through AMPK signaling. Consistent with CYP1A1 knockdown, pharmacologic reduction of CYP1A1 levels by the phytopolyphenol carnosol also correlated with impaired proliferation and induced AMPK phosphorylation. These results indicate that reduction of basal CYP1A1 expression is critical for inhibition of proliferation, which is not affected by α-naphthoflavone-mediated inhibition of CYP1A1 activity nor modulated by AhR silencing. This study supports the notion that CYP1A1 promotes breast cancer proliferation and survival, at least in part, through suppression of AMPK signaling and that reduction of CYP1A1 levels is a potential strategy for breast cancer therapeutics.