The effect of fish oil (FO) treatment on high‐fat (HF) diet‐induced obesity and metabolic syndrome was addressed by analyzing dysfunctions in cells of different adipose depots. For this purpose, mice were initially induced to obesity for 8 weeks following a treatment with FO containing high concentration of EPA compared to DHA (5:1), for additional 8 weeks (by gavage, 3 times per week). Despite the higher fat intake, the HF group showed lower food intake but higher body weight, glucose intolerance and insulin resistance, significant dyslipidemia and increased liver, subcutaneous (inguinal‐ING) and visceral (retroperitoneal‐RP) adipose depots mass, accompanied by adipocyte hypertrophy and decreased cellularity in both adipose tissue depots. FO treatment reversed all these effects, as well as it improved the metabolic activities of isolated adipocytes, such as glucose uptake and lipolysis in both depots, and de novo synthesis of fatty acids in ING adipocytes. HF diet also significantly increased both the pro and anti‐inflammatory cytokines expression by adipocytes, while HF + FO did not differ from control group. Collectively, these data show that the concomitant administration of FO with the HF diet is able to revert metabolic changes triggered by the diet‐induced obesity, as well as to promote beneficial alterations in adipose cell activities. The main mechanism underlying all systemic effects involves direct and differential effects on ING and RP adipocytes. The manuscript show relevant effects of fish oil (FO) treatment reversing both the metabolic syndrome induced by high‐fat diet (HF) and the changes in subcutaneous (inguinal ‐ING) and visceral (retroperitoneal ‐RP) adipocytes, such as glucose uptake, lipolysis, and lipogenesis. The major findings of our study is that the FO treatment reverses the deleterious effects caused by the excessive intake of a HF diet, and the mechanism involves, at least in part, direct and differential effects on ING and RP adipocytes. Altogether, these effects prevented adipocyte hypertrophy caused by HF diet and reflected in decreased adiposity. Additionally, FO treatment decreased the gene and protein expression of inflammation‐related factors. Thus, the work provided important evidence concerning the effectiveness of a low‐cost agent (as the FO) to treat the metabolic and inflammatory disorders triggered by obesity, which causes thousands of deaths annually and burdens the public coffers.