The stimulation of fat thermogenesis and modulation of the gut microbiota are promising therapeutic strategies against obesity. Zeaxanthin (ZEA), a carotenoid plant pigment, has been shown to prevent various diseases; however, the therapeutic mechanism for obesity remains unclear. Herein, whether ZEA improves obesity by activating the β3-adrenergic receptor (β3-AR) to stimulate white adipose tissue (WAT) thermogenesis and modulating the gut microbiota was investigated. C57BL6/N mice were fed a high-fat diet (HFD) supplemented with ZEA for 22 weeks. ZEA treatment reduced body weight, fat weight, adipocyte hypertrophy, liver weight, and lipid deposition, and improved dyslipidaemia, serum GPT, GOT, leptin, and irisin levels, glucose intolerance, and insulin resistance in HFD-fed mice. Mechanistically, ZEA treatment induced the expression of β3-AR and thermogenic factors, such as PRDM16, PGC-1α, and UCP1, in inguinal WAT (iWAT) and brown adipose tissue. ZEA treatment stimulated iWAT thermogenesis through the synergistic cooperation of key organelles, which manifested as an increased expression of lipid droplet degradation factors (ATGL, CGI-58 and pHSL), mitochondrial biogenesis factors (Sirt1, Nrf2, Tfam, Nampt and Cyt-C), peroxisomal biogenesis factors (Pex16, Pex19 and Pmp70), and β-oxidation factors (Cpt1, Cpt2, Acadm and Acox1). The thermogenic effect of ZEA was abolished by β3-AR antagonist (SR59230A) treatment. Additionally, dietary supplementation with ZEA reversed gut microbiota dysbiosis by regulating the abundance of Firmicutes, Clostridia, Proteobacteria, and Desulfovibrio, which were associated with the thermogenesis- and obesity-associated indices by Spearman's correlation analysis. Functional analysis of the gut microbiota indicated that ZEA treatment significantly enriched the lipid metabolism pathways. These results demonstrate that ZEA is a promising multi-target functional food for the treatment of obesity by activating β3-AR to stimulate iWAT thermogenesis, and modulating the gut microbiota.