Objective To investigate the mechanism of formononetin regulating the heat production of brown adipocytes via decoupling protein 1 (UCP1). Methods The brown preadipocytes was isolated from wild-type (WT) C57BL/6J mice and differentiated into mature fat cells in vitro. Moreover, the mRNA levels of fatty acid binding protein 4 (FABP4) and adiponectin were detected by real-time quantitative PCR (RT-qPCR). To confirm formononetin could induce the expression of thermogenic genes, we first prepared WT mature brown adipocytes and treated them with DMSO and formononetin separately. The mRNA and protein levels of thermogenic genes, such as peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), peroxisome proliferators-activated receptor γ (PPARγ), UCP1 and iodothyronine deiodinase 2 (Dio2), were detected by RT-qPCR and Western blot analysis. To investigate the role of UCP1 in mediating differentiation of brown preadipocytes, Fabp4 and adiponectin mRNA levels were analyzed by RT-qPCR in WT and UCP1 mutation differentiated brown adipocytes. To determine cellular oxygen consumption, isolated WT and UCP1 mutation brown preadipocytes were plated in an XF24-well microplate and differentiated into mature brown adipocytes treated with formononetin or DMSO, followed by oxygen consumption rate (OCR) measurement using XF24 analyser. Results Both WT and UCP1 KO brown preadipocytes could be differentiated into adipocyte. The expression of thermogenic genes, including PGC-1α, Dio2, PPARγ and UCP1, induced by formononetin was similar in UCP1 KO adipocytes and WT cells. But the ability of formononetin to increase cellular respiration was inhibited in Ucp1 KO cells. Conclusion Formononetin mediated stimulation of thermogenesis and oxygen consumption via UCP1 in brown fat cells.