This study was designed to establish whether 3,3′-diindolylmethane (DIM) can inhibit cervical lesions, alter estrogen metabolism in favor of C-2 hydroxylation, and enhance immune function in the K14-HPV16 transgenic mouse model. Mice were bred, genotyped, implanted with E₂ pellets (0.25 mg/90-day release) under anesthesia, and divided into groups. Wild-type and transgenic mice were given either AIN76A diet alone or with 2,000 ppm DIM for 12 weeks. Blood and reproductive tracts were obtained. Blood was analyzed for estrogen metabolites and IFN-γ. The cervical transformation zone was sectioned and stained for histology. Estradiol C-2 hydroxylation and serum IFN-γ levels were significantly increased over controls in wild-type and transgenic mice receiving DIM. In wild-type mice without DIM, hyperplasia of the squamous epithelium was observed. Wild-type mice fed DIM displayed a normal thin epithelium. In transgenic mice without DIM, epithelial cell projections into the stroma (papillae) were present. An additional degree of nuclear anaplasia in the stratum espinosum was observed. Dysplastic cells were present. Transgenic mice fed DIM displayed some mild hyperplasia of the squamous epithelium. DIM increases estrogen C-2 hydroxylation in this model. Serum INF-γ was increased, indicating increased immune response in the DIM-fed animals. Histopathology showed a marked decrease in cervical dsyplasia in both wild-type and transgenic mice, indicating that DIM delays or inhibits the progression from cervical dysplasia to cervical cancer. Using the K14-HPV16 transgenic mouse model, we have shown that DIM inhibits the development of E6/E7 oncogene–induced cervical lesions. (Cancer Epidemiol Biomarkers Prev 2009;18(11):2957–64)