Tumour necrosis factor (TNF)-α impaired insulin induction on GLUT4 mRNA in foetal brown adipocytes, as demonstrated by quantitative RT-PCR and Northern blot. We have explored the hypothesis that some effects of TNF-α could be mediated by the generation of ceramide, since TNF-α treatment induced the production of ceramide in these primary cells. A short-chain ceramide analogue, C2-ceramide, precluded insulin-induced GLUT4 mRNA accumulation and GLUT4-chloramphenicol acetyltransferase (CAT) full promoter activation. Moreover, inhibition of the ceramide biosynthesis with fumonisin B, which inhibits ceramide synthase, completely restored insulin-induced GLUT4 mRNA and protein accumulation as well as GLUT4-CAT transactivation in the presence of TNF-α. In consequence, TNF-α-induced insulin resistance on glucose uptake was completely alleviated. In addition, TNF-α down-regulated insulin-induced CCAAT/enhancer binding protein (C/EBP)-alpha gene expression and DNA binding activity, but fumonisin B precludes these effects. Furthermore, co-transfection with a wild-type C/EBP-α construct transactivates GLUT4-CAT construct. Our results indicate that de novo ceramide produced by TNF-α-induced insulin resistance on GLUT4 gene expression in brown adipocytes by interfering C/EBP-α expression, a transcription factor essential for the expression of GLUT4.