Brown adipose tissue (BAT) activation and subcutaneous white fat browning are essential components of the thermogenic response to cold stimulus in mammals. microRNAs have been shown to regulate both processes in cis. Here, we identify miR-32 as a BAT-specific super-enhancer-associated miRNA in mice that is selectively expressed in BAT and further upregulated during cold exposure. Inhibiting miR-32 in vivo led to impaired cold tolerance, decreased BAT thermogenesis, and compromised white fat browning as a result of reduced serum FGF21 levels. Further examination showed that miR-32 directly represses its target gene Tob1, thereby activating p38 MAP kinase signaling to drive FGF21 expression and secretion from BAT. BAT-specific miR-32 overexpression led to increased BAT thermogenesis and serum FGF21 levels, which further promotes white fat browning in trans. Our results suggested miR-32 and Tob1 as modulators of FGF21 signaling that can be manipulated for therapeutic benefit against obesity and metabolic syndrome.