Excessive energy intake is the main cause of obesity, and stimulation of brown adipose tissue (BAT) and white adipose tissue (WAT) thermogenesis has emerged as an attractive tool for anti-obesity. Although miR-143 has been reported to be associated with BAT thermogenesis, its role remains unclear. Here, we found that miR-143 was highest expression in adipose tissue, especially in BAT. During short-term cold exposure or CL316,243 injected, miR-143 was markedly down-regulated in BAT and subcutaneous WAT (scWAT). Moreover, knockout of miR-143 (KO) increases the body temperature of mice upon cold exposure, which may be due to the increased thermogenesis of BAT and scWAT. More importantly, supplement of miR-143 in BAT of KO mice can inhibit the increase of body temperature in KO mice. Mechanistically, spleen tyrosine kinase (SYK) was revealed for the first time as a new target of miR-143, and deletion of miR-143 facilitates fatty acid uptake in BAT. In addition, we found that brown adipocytes can promote fat mobilization of white adipocytes, and miR-143 may participate in this process. Meanwhile, we demonstrate that inactivation of AC9 in BAT inhibits thermogenesis through AC9-PKA-AMPK-CREB-UCP1 signaling pathway. Overall, our results reveal a novel function of miR-143 on thermogenesis, and a new functional link of the BAT and WAT.