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Purpose: To analyze the effects of a single garlic (Allium sativum) oil (SGO) on the activation of regulatory T cells and anti-inflammatory cytokines IL-10 and TGF-β in mice fed a high-fat diet (HFD). Methods: The study was conducted with 24 BALB/c male mice divided into six groups consisting of four mice each, i.e., control group (non-HFD, no treatment), HFD group without treatment, HFD administered with simvastatin, and three HFD groups administered SGO doses of 12.5, 25.0 and 50.0 mg/kg, respectively, and continuously treated (with SGO) for 45 days. The relative number of regulatory T cells, IL-10 and TGF β were measured using flow cytometry. Results: HFD decreased the expression of regulatory T cells, and the production of IL-10 and TGF-β compared to the control group. SGO (50 mg/kg) significantly enhanced the activation of regulatory T cells and production of TGF-β in mice fed high-fat diet compared to simvastatin group. The dose of 25 mg/kg SGO significantly increased the level of IL-10 in mice fed a HFD.