This study is designed to evaluate whether the PEGylated conjugated linoleic acid (PCLA) as the pro-drug can have favorable stability, bioavailability, and anti-adipogenic activity in 3T3-L1 cells for anti-obesity when compared with conjugated linoleic acid (CLA) itself. The CLA was simply coupled to poly(ethylene glycol) (PEG) at the melting state without solvents or catalysts through ester linkages between the carboxylic group of CLA and the hydroxyl group of PEG. To confirm of PCLA as the pro-drug, CLA release from PCLA was investigated by using high-performance liquid chromatographic (HPLC), showing that CLA release from PCLA was almost 90% in a nearly continuous fashion over the next 75h. Apoptosis was promoted by both CLA- and PCLA-treatments with increasing concentrations. However, the level of cell apoptosis induced by PCLA was lower than that induced by CLA owing to the biocompatible and hydrophilic properties of PEG. Moreover, the PCLA decreased glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 cells by acting upon major adipocyte marker proteins such as PPARgamma2, C/EBPalpha, and aP2 modulators. Furthermore, either CLA or PCLA stimulated basal, but not isoproterenol-sensitive, lipolysis in our cell model, suggesting that both CLA and PCLA may stimulate lipolysis via hormone sensitive lipase (HSL)-independent mechanisms. These results suggest that the PCLA may prove to be a stable pro-drug to control the deposition of fat in the human body, and that the anti-adipogenic effect of the PCLA on 3T3-L1 cells will offer a challenging approach for anti-obesity.