Scientific research is constantly working to find new molecules that are effective in preventing excessive accumulation of body fat. The aim of the present work was to assess the potential agonism on PPARα and PPARγ of a conjugated linolenic acid (CLNA) isomer mixture, consisting of two CLNA isomers (cis-9,trans-11,cis-15 and cis-9,trans-13,cis-15). Secondly, we aimed to analyze the effects of this mixture on triacylglycerol accumulation in 3T3-L1 mature adipocytes. Luciferase transactivation assay was used to analyze whether the CLNA mixture activated PPARs. The expression of several enzymes and transcriptional factors involved in the main metabolic pathways that control triacylglycerol accumulation in adipocytes was assessed by real time RT-PCR in 3T3-L1 adipocytes treated for 20 h with the CLNA mixture. The mixture activated PPRE in cells with PPARα receptor over-expression, but not those with PPARγ over-expression. Decreased triacylglycerol was found in treated adipocytes. The lowest dose (10 μM) increased HSL expression and the highest dose (100 μM) increased ATGL gene expression. The other genes analyzed remained unchanged. The hypothesis of an anti-obesity action of the analyzed CLNA mixture, based on increased lipid mobilization in adipose tissue, can be proposed.