Extensive studies have shown that the increasing brown adipose tissue (BAT) mass/activity possesses a strong ability to prevent obesity and its related complications. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signal pathway is known to play a role in adipocyte differentiation and development. However, its impact on thermogenic properties of mature brown adipocytes has not yet been clarified. Nifuroxazide (NFX), a potent inhibitor of STAT3, has received widespread attention due to its alternative anti-tumor and anti-inflammatory effects. Herein, we report that NFX induces lipolysis with subsequent downregulation of ACC alpha and FAS, while ATGL and pHSL levels are elevated in mature brown adipocytes. Furthermore, NFX treatment promotes the mitochondrial respiration of mature brown adipocytes, as evidenced by increased expression of thermogenic transcriptional factors and mitochondrial content. In addition, it also alleviates the IL6 and TNF alpha inhibition on brown thermogenic programming via suppressing the STAT3/NF-kappa B/IL-6 signaling pathway. In general, these findings suggest that the blockade of the JAK/STAT3 pathway by NFX has a prothermogenic effect on mature brown adipocytes which opens new perspectives for NFX repurposing and potential therapeutic route to counteract obesity and related metabolic disorders.