Conjugated linoleic acid (CLA) is reported to have health benefits, including reduction of body fat. Previous studies have shown that brown adipose tissue (BAT) is particularly sensitive to CLA supplemented diet-feeding. Most of them use mixtures containing several CLA isomers, mainly cis-9, trans-11 and trans-10, cis-12 in equal concentration. Our aim was to characterize the separate effects of both CLA isomers on thermogenic capacity in cultured brown adipocytes. The CLA isomers showed opposite effects. Hence, on the one hand, trans-10, cis-12 inhibited UCP1 induction by noradrenaline (NA), and produced a decrease in leptin mRNA levels. These effects were associated with a blockage of C/EBP and PPAR2 mRNA expression. On the other hand, cis-9, trans-11 enhanced the UCP1 elicited by NA, an effect earlier reported for polyunsaturated fatty acids and also observed here for LA. These findings could explain, at least in part, the effects observed in vivo, when feeding a CLA-mixture supplemented-diet as a result of the combined action of CLA isomers: reduction of adipogenesis and defective BAT thermogenesis which could be through trans-10, cis-12, and enhanced UCP1 thermogenic capacity through cis-9, trans-11.