The trans-10,cis-12 isomer of conjugated linoleic acid (CLA) has been shown to reduce body fat gain in mice. However, the underlying molecular mechanism is not well characterized. Here we report evidence that trans-10,cis-12 (t10c12) CLA inhibits preadipocyte differentiation. Treating differentiating 3T3-L1 preadipocytes with t10c12 CLA and conjugated nonadecadienoic acid (CNA, a 19-carbon CLA cognate) resulted in decreased intracellular triglyceride accumulation and mRNA levels of the adipogenic gene fatty acid synthase and adipocyte lipid binding protein. T10c12 CLA and CNA also reduced protein levels of adipocyte transcription factors, peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Similarly, CLA reduced body fat gain and significantly inhibited the expression of PPAR gamma and its downstream target lipoprotein lipase in mouse adipose tissue. These observations indicate that CLA decreases body fat gain in part by inhibiting the differentiation of preadipocytes.