Conjugated linoleic acid (CLA) is widely used as a "nutraceutical" for weight loss. CLA has anticancer effects in preclinical models, and we demonstrated in vitro that this can be attributed to the suppression of fatty acid (FA) synthesis. We tested the hypothesis that administration of CLA to breast cancer patients would inhibit expression of markers related to FA synthesis in tumor tissue, and that this would suppress tumor proliferation. Women with Stage I-III breast cancer were enrolled into an open label study and treated with CLA (1:1 mix of 9c,11t- and 10t,12c-CLA isomers, 7.5 g/d) for ≥ 10 days before surgery. Fasting plasma CLA concentrations measured pre- and post-CLA administration, and pre/post CLA tumor samples were examined by immunohistochemistry for Spot 14 (S14), a regulator of FA synthesis, FA synthase (FASN), an enzyme of FA synthesis, and lipoprotein lipase (LPL), the enzyme that allows FA uptake. Tumors were also analyzed for expression of Ki-67 and cleaved caspase 3. 24 women completed study treatment, and 23 tumors were evaluable for the primary endpoint. The median duration of CLA therapy was 12 days, and no significant toxicity was observed. S14 expression scores decreased (p = 0.003) after CLA administration. No significant change in FASN or LPL expression was observed. Ki-67 scores declined (p = 0.029), while cleaved caspase 3 staining was unaffected. Decrements in S14 or Ki-67 did not correlate with fasting plasma CLA concentrations at surgery. Breast tumor tissue expression of S14, but not FASN or LPL, was decreased after a short course of treatment with 7.5 g/day CLA. This was accompanied by reductions in the proliferation index. CLA consumption was well-tolerated and safe at this dose for up to 20 days. Overall, CLA may be a prototype compound to target fatty acid synthesis in breast cancers with a "lipogenic phenotype".