Murine 3T3-L1 adipocytes share many similarities with primary fat cells and represent a reliable in vitro model of adipogenesis. The aim of this study was to probe the effect of S-nitrosoglutathione (GSNO) on adipocyte differentiation. Adipogenesis was induced with a mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the absence and presence of increasing GSNO concentrations. Biochemical analysis after 7 days of differentiation showed a prominent anti-adipogenic effect of GSNO which was evident as reduced cellular triglycerides and total protein content as well as decreased mRNA and protein expression of late transcription factors (e.g. peroxisome proliferator activated receptor γ) and markers of terminal differentiation (e.g. leptin). By contrast, the nitrosothiol did not affect mRNA and protein expression of CCAAT/enhancer-binding protein β (C/EBPβ), which represents a pivotal early transcription factor of the adipogenic cascade. Differentiation was also inhibited by the NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. Biotin switch experiments showed significantly increased S-nitrosation of C/EBPβ variants indicating that posttranslational S-nitrosative modification of this transcription factor accounts for the observed anti-adipogenic effect of NO. Our results suggest that S-nitrosation might represent an important physiological regulatory mechanism of fat cell maturation.