Prdm16 determines the bidirectional fate switch of skeletal muscle/brown adipose tissue (BAT) and regulates the thermogenic gene program of subcutaneous white adipose tissue (SAT) in mice. Here we show that miR-133a, a microRNA that is expressed in both BAT and SATs, directly targets the 3' UTR of Prdm16. The expression of miR-133a dramatically decreases along the commitment and differentiation of brown preadipocytes, accompanied by the upregulation of Prdm16. Overexpression of miR-133a in BAT and SAT cells significantly inhibits, and conversely inhibition of miR-133a upregulates, Prdm16 and brown adipogenesis. More importantly, double knockout of miR-133a1 and miR-133a2 in mice leads to elevations of the brown and thermogenic gene programs in SAT. Even 75% deletion of miR-133a (a1(-/-)a2(+/-) ) genes results in browning of SAT, manifested by the appearance of numerous multilocular UCP1-expressing adipocytes within SAT. Additionally, compared to wildtype mice, miR-133a1(-/-)a2(+/-) mice exhibit increased insulin sensitivity and glucose tolerance, and activate the thermogenic gene program more robustly upon cold exposure. These results together elucidate a crucial role of miR-133a in the regulation of adipocyte browning in vivo.