Brown adipose tissue (BAT) plays a key role in energy expenditure through its specialized thermogenic function. Therefore, BAT activation may help prevent and/or treat obesity. Interestingly, subcutaneous white adipose tissue (WAT) also has the ability to differentiate into brown-like adipocytes and may potentially contribute to increased thermogenesis. We have previously reported that eicosapentaenoic acid (EPA) reduces high-fat (HF)-diet-induced obesity and insulin resistance in mice. Whether BAT mediates some of these beneficial effects of EPA has not been determined. We hypothesized that EPA activates BAT thermogenic program, contributing to its antiobesity effects. BAT and WAT were harvested from B6 male mice fed HF diets supplemented with or without EPA. HIB 1B clonal brown adipocytes treated with or without EPA were also used. Gene and protein expressions were measured in adipose tissues and H1B 1B cells by quantitative polymerase chain reaction and immunoblotting, respectively. Our results show that BAT from EPA-supplemented mice expressed significantly higher levels of thermogenic genes such as PRDM16 and PGC1α and higher levels of uncoupling protein 1 compared to HF-fed mice. By contrast, both WATs (subcutaneous and visceral) had undetectable levels of these markers with no up regulation by EPA. HIB 1B cells treated with EPA showed significantly higher mRNA expression of PGC1α and SIRT2. EPA treatment significantly increased maximum oxidative and peak glycolytic metabolism in H1B 1B cells. Our results demonstrate a novel and promising role for EPA in preventing obesity via activation of BAT, adding to its known beneficial anti-inflammatory effects.