Docosahexaenoic acid (DHA; C22:6n−3) depresses mammary carcinoma proliferation and growth in cell culture and in animal models. The current study explored the role of interrupting bioenergetic pathways in BT‐474 and MDA‐MB‐231 breast cancer cell lines representing respiratory and glycolytic phenotypes, respectively and comparing the impacts of DHA with a non‐transformed cell line, MCF‐10A. Metabolic investigation revealed that DHA supplementation significantly diminished the bioenergetic profile of the malignant cell lines in a dose‐dependent manner. DHA enrichment also resulted in decreases in hypoxia‐inducible factor (HIF‐1α) total protein level and transcriptional activity in the malignant cell lines but not in the non‐transformed cell line. Downstream targets of HIF‐1α, including glucose transporter 1 (GLUT 1) and lactate dehydrogenase (LDH), were decreased by DHA treatment in the BT‐474 cell line, as well as decreases in LDH protein level in the MDA‐MB‐231 cell line. Glucose uptake, total glucose oxidation, glycolytic metabolism, and lactate production were significantly decreased in response to DHA supplementation; thereby enhancing metabolic injury and decreasing oxidative metabolism. The DHA‐induced metabolic changes led to a marked decrease of intracellular ATP levels by 50% in both cancer cell lines, which mediated phosphorylation of metabolic stress marker, AMPK, at Thr172. These findings show that DHA contributes to impaired cancer cell growth and survival by altering cancer cell metabolism, increasing metabolic stress and altering HIF‐1α‐associated metabolism, while not affecting non‐transformed MCF‐10A cells. This study provides rationale for enhancement of current cancer prevention models and current therapies by combining them with dietary sources, like DHA. © 2014 Wiley Periodicals, Inc.