Adipogenesis is the overall process of adipocyte differentiation. With a positive energy balance, intracellular accumulation of triglyceride increases through the generation of functional adipocytes. This occurs with adipogenesis of undifferentiated preadipocytes. Citrullus colocynthis (CC), a member of the Cucurbitaceae family, is a desert viny plant native to North Africa. The effect of ethanolic seed extracts of colocynth (SCEE) on adipogenesis was investigated using 3T3-L1 preadipocytes. Cell viability was measured using the MTT assay; triglycerides were stained with Oil Red O and adipogenesis-related gene expressions were quantified using qRT-PCR. Results showed that SCEE helps to inhibit intracellular triglyceride accumulation during adipogenesis without affecting cell viability. Likewise, SCEE not only showed anti-adipogenic activities, essentially during the early stage, but their effects during the middle and late stages were very low. These effects have been confirmed by the down-regulation of C/EBP beta and C/EBP delta at 24, 48 and 72 h. In addition, SCEE treatment in 3T3-L1 cells induced down-regulation of the transcription factors CCAAT/enhancer binding protein a (C/EBP alpha), peroxisome proliferator activated receptor gamma (PPAR gamma), and sterol regulatory element-binding protein 1c (SREBP-1c) and their target genes. UPLC-ESI-MS/MS profiling showed 10 bioactive compounds including quinic acid, isovitexin, scoparin, vitexin-2 ''-O-rhamnoside, reserpine, digitoxin, triprolidine, naringenin, linoleic acid and oleic acid. Thus, these results suggested that the seeds of colocynth may have beneficial health effects.