Genistein has beneficial effects on glucose metabolism and adipose tissue browning which paralleled with gut microbiota alterations. However, the causality is unclear. This study aims to evaluate whether genistein could ameliorate metabolism and activate the browning program and whether gut microbiota is indispensable for these alterations. We examined the effect of 10-week genistein gavage (30 mg kg-1 d-1) on glucose metabolism in C57BL/6J mice. Cecal contents were collected for 16s rRNA sequencing. The mRNA of browning markers was quantified in adipose tissues. Antibiotic administration was used to deplete gut microbiota. The lean mice with a normal control diet and genistein exhibited better glucose tolerance and higher expression of UCP1 and PGC1α in white fat compared with those without genistein. Markedly enriched Blautia, Ruminiclostridium_5, and Ruminiclostridium_9 in genistein-treated mice were significantly correlated with browning markers and glucose tolerance. In obese mice, genistein alleviated the detrimental effects of a high-fat diet on glucose homeostasis and increased UCP1 and PGC1α expression in brown fat. Obvious increases in Ruminiclostridium, Rikenella, and Clostridium_sensu_stricto_1 by genistein were associated with metabolic improvement. However, depleting gut microbiota abolished these benefits. Overall, our findings indicated that gut microbiota contributed to enhanced glucose metabolism and adipose tissue browning of genistein, providing a promising target for metabolic health protection.