Considering that melatonin has been implicated in body weight control, this work investigated whether this effect involves the regulation of adipogenesis. 3T3‐L1 preadipocytes were induced to differentiate in the absence or presence of melatonin (10⁻³ m). Swiss‐3T3 cells ectopically and conditionally (Tet‐off system) over‐expressing the 34 kDa C/EBPβ isoform (Swiss‐LAP cells) were employed as a tool to assess the mechanisms of action at the molecular level. Protein markers of the adipogenic phenotype were analyzed by Western blot. At 36 hr of differentiation of 3T3‐L1 preadipocytes, a reduction of PPARγ expression was detected followed by a further reduction, at day 4, of perilipin, aP2 and adiponectin protein expression in melatonin‐treated cells. Real‐time PCR analysis also showed a decrease of PPARγ (60%), C/EBPα (75%), adiponectin (30%) and aP2 (40%) mRNA expression. Finally, we transfected Swiss LAP cells with a C/EBPα gene promoter/reporter construct in which luciferase expression is enhanced in response to C/EBPβ activity. Culture of such transfected cells in the absence of tetracycline led to a 2.5‐fold activation of the C/EBPα promoter. However, when treated with melatonin, the level of C/EBPα promoter activation by C/EBPβ was reduced by 50% (P = 0.05, n = 6). In addition, this inhibitory effect of melatonin was also reflected in the phenotype of the cells, since their capacity to accumulate lipids droplets was reduced as confirmed by the poor staining with Oil Red O. In conclusion, melatonin at a concentration of 10⁻³ m works as a negative regulator of adipogenesis acting in part by inhibiting the activity of a critical adipogenic transcription factor, C/EBPβ.