Background: In vitro studies evidenced antitumor effects of omega-3 polyunsaturated fatty acids ([n−3] PUFAs), but their effects on prostate cancer (PCa) remain controversial in epidemiological studies. Here we investigated whether an (n−3) PUFA-enriched diet affects tumor progression in transgenic adenocarcinoma of the mouse prostate (TRAMP), at early (12 weeks age) and advanced stages (20 weeks age).Methods: TRAMP mice were fed with standard rodent diet (C12, C20) or (n−3) PUFA-enriched diet containing 10% fish oil (T12, T20). A group of 8 weeks age animals fed standard diet was also used for comparison (C8). The ventral prostate was processed for histopathological and immunohistochemical analyses and serum samples submitted to biochemical assays.Results: At early stages, (n−3) PUFA increased the frequency of normal epithelium (3.8-fold) and decreased the frequency of high-grade intraepithelial neoplasia (3.3-fold) and in situ carcinoma (1.9-fold) in the gland, maintaining prostate pathological status similar to C8 group. At advanced stages, 50% of the animals developed a large primary tumor in both C20 and T20, and tumor weight did not differ (C20: 2.2 ± 2.4; T20: 2.8 ± 2.9 g). The ventral prostate of T12 and of T20 animals that did not develop primary tumors showed lower cell proliferation, tissue expressions of androgen (AR) and glucocorticoid (GR) receptors, than their respective controls. For these animals, (n−3) PUFA also avoided an increase in the number of T-lymphocytes, collagen fibers, and αSMA immunoreactivity, and preserved stromal gland microenvironment. (n−3) PUFA also lowered serum triglycerides and cholesterol, regulating the lipid metabolism of TRAMP mice.Conclusions: (n−3) PUFAs had a protective effect at early stages of PCa, delaying tumor progression in TRAMP mice, in parallel with reductions in cell proliferation, AR, and GR and maintenance of the stromal compartment of the gland. However, (n−3) PUFAs did not prevent the development of primary tumors for the T20 group, reinforcing the need for further investigation at advanced stages of disease.