The putative peroxisome proliferator-activated receptor (PPAR) α ligand, conjugated linoleic acid (CLA) induced cytoplasmic lipid droplet (LD) formation in H4IIE rat hepatoma cells. Currently, the mechanism(s) by which CLA isomers affects hepatic LD formation is unclear. We have investigated the role of PPARα and fatty acid (FA) activation in the regulation of hepatic LD formation induced by CLA isomers [cis-9,trans-11 (c9,t11), trans-10,cis-12 (t10,c12)] and linoleic acid (LA) in an in vitro model of lipid accumulation. Dose response of c9,t11 and t10,c12 CLA isomers as well as LA in quiescent H4IIE cells was assessed by Oil Red O staining and subsequent quantification after 24 hours. LD formation was induced by the CLA isomers similar to LA in a dose-dependent manner. However, treatment with the acyl CoA synthetase (ACS) inhibitor, triacsin C, resulted in significantly reduced LD formation. A similar reduction in lipid accumulation was observed with the PPARα activator, Wy14643. Furthermore, CLA isomers promoted H4IIE viability at 60 µM but decreased viability at a higher dose of 180 µM. To further understand the role of PPARα in hepatic steatosis, we studied the level and phosphorylation of PPARα in livers of male lean and fa/fa Zucker rats fed either a control diet or fa/fa Zucker rats fed a CLA isomer (0.4% wt/wt c9,t11 or 0.4% wt/wt t10,c12) diet for 8 weeks. Immunoblotting results showed that only the t10,c12 CLA isomer significantly reduced phospho-PPARα S21 compared to the lean control (ln Ctl) and it was associated with a significant increase in the phosphorylation of p38 mitogen activated protein kinase (MAPK).These changes were not observed with the c9,t11 CLA isomer. Taken together, we have shown that CLA isomers directly induce LD formation in quiescent H4IIEs by activation of the lipid storage pathway which was significantly reduced by triacsin C or Wy14643. Also, we demonstrate for the first time that only the t10,c12 CLA isomer significantly reduced PPARα phosphorylation while it increased p38 MAPK phosphorylation. These results indicate that the anti-steatotic effects of the t10,c12 CLA isomer is associated with changes in PPARα phosphorylation and thereby its activity in a MAPK-independent manner.