Purpose: HER-2/neu overexpression is a molecular subtype of breast cancer associated with poorer clinical outcome. The association of low-grade inflammation with increased risk for cancer and correlation of HER-2/neu overexpression and immune cell infiltration in human breast tumors suggest that an inflammatory mammary microenvironment is critical to early events in HER-2/neu carcinogenesis. Of interest are nutritional, complementary interventions that may suppress mammary gland inflammation, such as dietary ω-3 fatty acids. This study is testing the hypothesis that dietary fat content can modulate inflammation during early stages of HER-2/neu mammary carcinogenesis, with inhibition of mammary macrophage infiltration and activation by fish oil/ω-3 fatty acids relative to corn oil/ω-6 fatty acids. Methods: Female MMTV-HER-2/neu transgenic mice were fed semi-purifed diets with 25% energy as menhaden fish oil or corn oil x 29 weeks or corn oil x 19 weeks then fish oil x 10 weeks. Mammary glands were harvested at 35 weeks for F480, CD3, and LY6G immunostaining to identify macrophages, T cells, and granulocytes, respectively. A novel MMTV-HER-2/neu;c-fms-YFP double transgenic mouse model was generated to facilitate isolation of mammary gland macrophages. HER-2/neu;c-fms-YFP and control c-fms-YFP mice were fed fish or corn oil based diets x 19 weeks. YFP+ cells were sorted using high-speeed digital FACS and RNA isolated from the enriched cells. Differential gene expression based on diet and genotype was determined by Affymetrix microarray. Results: Diets were well tolerated by the mice without toxicity. By immunostaining, MMTV-HER-2/neu mammary glands of fish oil fed mice had a lower number of macrophages/area of mammary duct relative to the corn oil fed mice (p<0.0001). Mice treated first with corn oil then changed to menhaden oil also had a lower number of macrophages/duct area compared to the corn oil fed mice, without significant differences from the fish oil group. CD3 and LY6G immunostaining did not reveal a similar pattern. In MMTV-HER-2/neu;c-fms-YFP and c-fms-YFP control mice, the mean percentage of YFP+ cells in thoracic and inguinal mammary fat pads and abdominal fat did not differ significantly by diet or genotype. Gene expression analyses by Affymetrix microarray indicate that the corn oil diets elicited a pro-inflammatory gene signature in mammary macrophages from HER-2/neu(+/+) mice relative to all other genotypes and dietary conditions. Conclusions: Dietary ω-3 versus ω-6 fatty acids may modulate mammary macrophage localization patterns without affecting the overall number of infiltrating macrophages. Dietary fish oil/ω-3 fatty acids may inhibit pro-inflammatory signaling mediated by mammary macrophages in early stages of HER-2/neu carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5426. doi:1538-7445.AM2012-5426