Although fish oil has hypolipidemic and antiatherosclerotic properties, the potential for white adipose tissue (WAT) to mediate these effects has not been studied. LDL-receptor deficient (LDLR-/-) mice were fed high fat, olive oil-containing diets supplemented with additional olive oil or with fish oil for 12 wk. Fish oil feeding significantly reduced plasma lipid levels. In contrast, lipid storage in WAT was increased in fish oil-fed mice as evidenced by increased total fat (P < 0.05) and perigonadal WAT mass (P < 0.05), increased cholesterol storage (P < 0.001), and adipocyte hypertrophy. Despite increased adipose tissue mass, WAT-specific inflammation and insulin sensitivity were improved (P < 0.05), concomitant with reduced macrophage infiltration. Furthermore, fish oil increased WAT and plasma levels of adiponectin. In addition, fish oil feeding decreased the formation of proinflammatory F2- isoprostanes, markers of oxidative stress (P < 0.05). The increased WAT lipid storage in fish oil-fed mice was associated with reduced lipid accumulation in liver (P < 0.05) and decreased atherosclerotic lesion area (P < 0.05). Taken together, these data highlight the specific role of WAT in regulating dietary fish oil-mediated improvement in systemic lipid homeostasis and atherosclerosis.