In this study, we hypothesized that trans(t)8,cis(c)10 conjugated linoleic acid (CLA) isomer possesses anti-adipogenic properties in resemblance to its structural analog, the t10,c12 CLA isomer. After chemical synthesis and purification of the t8,c10 CLA isomer in our laboratory, we report, for the first time, its individual action on the lipid metabolism of 3T3-L1 adipocytes. Cells were supplemented with 50 mM of linoleic acid (LA), t10,c12, or t8,c10 during 11 days of differentiation. Intracellular triacylglycerols (TAG) content was affected by fatty acid treatments. The t8,c10 CLA isomer reduced TAG content but in a lower magnitude, three times less, than the t10,c12 isomer. The supplementation with LA increased TAG storage thus reversing CLA isomers effect. The fatty acid profile of treated adipocytes showed that the t8,c10 CLA isomer incorporation was identical to the t10,c12. The mRNA expression of several genes involved in lipogenesis, lipolysis, fatty acid b-oxidation, and adipocytes differentiation underlying anti-adipogenic action was determined. The t10,c12 CLA isomer decreased gene expression levels related to lipogenesis but did not up-regulate HSL and PPARA associated with lipolysis and fatty acid b-oxidation, respectively. The t8,c10 CLA isomer increased SCD mRNA level without affecting delta 9 desaturase index-16, an indicator of SCD enzyme activity. Taken together, our results indicate that the t8,c10 CLA isomer, tested individually on lipid metabolism of 3T3-L1 adipocytes, is anti-adipogenic.Practical applications: New and more effective weight loss supplements are widely pursued facing the worldwide obesity epidemic. Over the last few decades, the anti-adipogenic properties of conjugated linoleic acid (CLA) have been studied and confirmed for the t10,c12 CLA isomer but, until now, there was no information on its structural analog, the t8,c10. This study provides novel insights on the putative anti-adipogenic ability of the t8,c10 CLA isomer exploiting potential molecular mechanisms of action in 3T3-L1 adipocytes.