Obesity is a metabolic disorder characterized by the central distribution of abdominal fat, hyperglycemia, hyperlipidemia, and hypertension. A high-fat diet can lead to overnutrition and directly trigger inflammation in adipose tissue. Regulatory T cells (Tregs) are essential negative regulators of inflammation. Soybean (Glycine max L.) has a variety of beneficial health. It contains isoflavones, particularly daidzein and genistein which can be transformed using microbial and physical stimuli to enhance bioactivity. The aim of this study was to analyze the effect of elicited soybean extract (ESE) on Treg activity in high fat-fructose (HFFD) mice. Twenty-eight female Balb/C mice were divided into seven groups: normal diet (ND) only, ND + ESE 104 mg/kg BW, HFFD only, HFFD + Simvastatin 2.8 mg/kg, HFFD + ESE 78 mg/kg BW, HFFD + ESE 104 mg/kg BW, and HFFD + ESE 130 mg/kg BW. The high fat-fructose diet was given over a period of 20 weeks, and ESE was administered orally per day after 20 weeks for four weeks. At week 24, the animals were sacrificed and the spleen was collected. Tregs were labeled as CD4 + CD25 + CD62L + and the relative Treg number was measured using flow cytometry. The HFFD treatment significantly decreased Treg number (p < 0.05) compared to a normal diet. The ESE treatment in HFFD mice could improve Treg numbers compared to HFFD mice. Our results suggest that ESE has potential to be used as a supplement to suppress chronic inflammation via increased Treg number.