Adiponutrin/PNPLA3 is a protein highly produced in adipose tissue whose expression is under tight nutritional regulation. It possesses lipogenic/lipolytic capacity and, although adiponutrin polymorphisms are related to obesity, its physiological role is not clear. To help clarify its role, we studied the effect of acute cold exposure on adiponutrin mRNA expression in different adipose tissues of lean/obese Zucker rats subjected to feeding/fasting/refeeding. The effect of cold on the expression of key lipogenic enzymes and on uncoupling protein-1 (UCP1) was evaluated in selected adipose depots. Adiponutrin mRNA levels were also determined in the adipose tissue of isoprenaline-treated rats and in cultured adipocytes treated with noradrenaline, isoprenaline and a selective β3-adrenoceptor (AR) agonist. Adiponutrin expression was strongly down-regulated by cold in the different adipose depots in lean animals, while this down-regulation was impaired in obese rats. Adiponutrin pattern of expression in response to cold correlated positively with that of the lipogenic enzymes and negatively with UCP1 expression. Acute intraperitoneal administration of isoprenaline also produced a decrease in adiponutrin expression in adipose tissue. In vitro data suggest that adiponutrin's inhibitory effect could be mediated, at least in part, by the sympathetic system via β1/β2-AR. In addition, improvement in metabolic parameters related to obesity in cold-exposed animals was related to an improvement in adiponutrin nutritional regulation. Thus, cold inhibition of adiponutrin expression in adipose tissue (which correlates with the response of lipogenic enzymes) supports a physiological role for this protein in lipogenesis. Moreover, alterations in adiponutrin expression and regulation in adipose tissue are related to obesity.