Breast cancer is the leading cause of death in women among all cancer types. Obesity is one of the factors that promote progression of breast cancer, especially in post-menopausal women. Increasingly, adipose tissue is recognized for its active role in the tumor microenvironment. We hypothesized that adipocytes conditioned medium can impact breast cancer progression by increasing inflammatory cytokines production by cancer cells, and subsequently increasing their motility. By contrast, eicosapentaenoic acid (EPA), an anti-inflammatory n-3 polyunsaturated fatty acid, reduces adipocyte-secreted inflammatory factors, leading to reduced cancer cell motility. To test these hypotheses, we investigated the direct effects of EPA on MCF-7 and MDA-MB-231 breast cancer cells and the effects of conditioned medium from 3 T3-L1 or human mesenchymal stem cells (HMSC)-derived adipocytes treated with or without EPA supplementation on breast cancer cells. We observed that conditioned medium from HMSC-derived adipocytes significantly increased mRNA transcription levels of cancer-associated genes such as FASN, STAT3 and cIAP2, while EPA-treated HMSC-derived adipocytes significantly reduced mRNA levels of these genes. However, direct EPA treatment significantly reduced mRNA content of these tumor-associated markers (FASN, STAT3, cIAP-2) only in MDA-MB-231 cells not in MCF-7 cells. Conditioned medium from EPA-treated 3 T3-L1 adipocytes further decreased inflammation, cell motility and glycolysis in cancer cells. Our data confirms that adipocytes play a significant role in promoting breast cancer progression and demonstrates that EPA-treated adipocytes reduced the negative impact of adipocyte-secreted factors on breast cancer cell inflammation and migration.