Obesity and associated complications are becoming a pandemic. Inhibiting adipogenesis is an important intervention for the treatment of obesity. Despite intensive investigations, numerous mechanistic aspects of adipogenesis remain unclear, and many potential therapeutic targets have yet to be discovered. Transcriptomics and lipidomics approaches were used to explore the functional genes regulating adipogenic differentiation and the potential mechanism in OP9 cells and adipose-derived stem cells. In this study, we found that NADH:ubiquinone oxidoreductase subunit A6 (Ndufa6) participates in the regulation of adipogenic differentiation. Furthermore, we show that the effect of Ndufa6 is mediated through stearoyl-CoA desaturase 1 (Scd1) and demonstrate the inhibitory effect of a SCD1 inhibitor on adipogenesis. Our study broadens the understanding of adipogenic differentiation and offers NDUFA6-SCD1 as a potential therapeutic target for the treatment of obesity.