Browning of white adipose tissue (WAT) is currently considered a potential therapeutic strategy to treat diet-induced obesity. While some probiotics have protective effects against diet-induced obesity, the role of probiotics in adipose browning has not been explored. Here, we show that administration of the probiotic bacterium Lactobacillus amylovorus KU4 (LKU4) to mice fed a high-fat diet (HFD) enhanced mitochondrial levels and function, as well as the thermogenic gene program (increased Ucp1, PPARγ, and PGC-1α expression and decreased RIP140 expression), in subcutaneous inguinal WAT and also increased body temperature. Furthermore, LKU4 administration increased the interaction between PPARγ and PGC-1α through release of RIP140 to stimulate Ucp1 expression, thereby promoting browning of white adipocytes. In addition, lactate, the levels of which are elevated in plasma of HFD-fed mice following LKU4 administration, elicited the same effect on the interaction between PPARγ and PGC-1α in 3T3-L1 adipocytes, leading to a brown-like adipocyte phenotype that included enhanced Ucp1 expression, mitochondrial levels and function, and oxygen consumption rate. Together, these data reveal that LKU4 facilitates browning of white adipocytes through the PPARγ-PGC-1α transcriptional complex, at least in part by increasing lactate levels, leading to inhibition of diet-induced obesity.