Intestinal microflora is correlated with obesity, metabolic diseases and digestive tract dysfunctions that are closely related to human health. Nobiletin (NOB) is a dietary polymethoxylated flavonoid with protective effects and activities against oxidative stress, inflammation and cardiovascular disorders. However, the effect and molecular mechanism of NOB in regulating white fat deposition have not been explored. In this study, we reported that NOB administration attenuates weight gain and glucose tolerance in mice fed a high−fat diet (HFD). Additionally, NOB administration substantially restored lipid metabolic disorder and repressed the level of genes related to lipid metabolism in HFD−induced obese mice. The sequencing of 16S rRNA genes in fecal samples unveiled that NOB administration reversed HFD−induced intestinal microbiota composition, particularly in the relative abundances of Bacteroidetes and Firmicutes at the phylum and genus level. Furthermore, NOB supplementation significantly improved the indexes of Chao1 and Simpson and implied NOB can improve intestinal flora diversity in HFD−fed mice. Next, we used LEfSe analysis to explore biomarkers presented as a taxon in different groups. Compared to the HFD group, NOB treatment significantly diminished the proportion ofRuminococcaceae, Ruminiclostridium, Intesinimonas, OscillibacterandDesulfovibrio. Enriched metabolic pathways were predicted by Tax4Fun analysis and demonstrated that the lipid metabolic pathway is higher in the HFD + NOB group. More importantly, the correlation analysis demonstrated thatParabacteroideswas significantly positive andLactobacilluswas negatively related to both body weight and inguinal adipose tissue weight. Collectively, our data emphasized that NOB has the potential to attenuate obesity and confirmed a mechanism for gut microbiota that mediated the beneficial effect of NOB.