Cytochrome P450 1B1 (CYP1B1) catalyzes estrogen hydroxylation and activation of potential carcinogens. Here we explored the role of CYP1B1 in endometrial carcinogenesis. Immunohistochemical staining of endometrial carcinomas showed that CYP1B1 is up-regulated in endometrial cancers. To understand the functional significance of CYP1B1 up-regulation in endometrial cancers with regard to tumorigenesis, we used small interfering RNA-mediated approach to knockdown CYP1B1 in endometrial carcinoma cell line followed by functional assays. Further, to understand the molecular basis of the role of CYP1B1 in endometrial carcinomas, we profiled the expression of key pathway-specific genes and identified several components of cell cycle, apoptosis, and cell adhesion pathways that are potentially regulated by CYP1B1. CYP1B1 depletion in endometrial carcinoma cells leads to decreased cellular proliferation and induces G(0)-G(1) cell cycle arrest. Significantly, CYP1B1 knockdown leads to down-regulated expression of cyclin E1, S-phase kinase-associated protein 2 (SKP2), minichromosome maintenance complex component 4 (MCM4), and RAD51 and up-regulation of p27(Kip1). Also, we identified cyclin E-binding protein (CEBP1) as a novel CYP1B1 target. Attenuation of CYP1B1 expression in endometrial carcinoma cells induces apoptosis and increases expression of IFN-beta (IFNbeta), granzyme A (GRZA), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, CYP1B1 depletion decreased the invasive potential of the endometrial cancer cells and expression of melanoma cell adhesion molecule (MCAM). In conclusion, our data suggest that CYP1B1 up-regulation plays a crucial role in endometrial carcinogenesis by targeting multiple pathways. We speculate that CYP1B1 inhibition in endometrial carcinomas could be a useful therapeutic approach as it regulates several potential anticancer targets like cyclin E1, Skp2, and TRAIL.