Obesity is highly correlated with several metabolic disorders. Adipocyte differentiation is a key process in determining obesogenesis. 14-Deoxy-11,12-didehydroandrographolide (deAND) is a diterpenoid rich in Andrographis paniculata (Burm.f.) Nees., a herbal medicine commonly used to treat colds, infections, and liver diseases. We investigated whether deAND inhibits the adipogenesis of 3T3-L1 cells and the underlying mechanisms. We found that deAND (0–15μM) dose-dependently inhibits the mRNA and protein expression of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein 1c, fatty acid synthase, and stearoyl-CoA desaturase-1. Cellular lipid accumulation was decreased by deAND, and the early phase of adipocyte differentiation was critical for this inhibition. Immunoblotting revealed that deAND attenuated differentiation medium–induced protein kinase A (PKA) and cAMP response element-binding protein (CREB) activation, which leads to down-regulating C/EBPβ transcription. Moreover, deAND inhibited ERK- and GSK3β-mediated C/EBPβ transcriptional activity. Flow cytometry analysis showed that deAND impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the G0/G1 phase, while the expression of cyclin D1, cyclin E, CDK6, and CDK2 was attenuated. deAND increased the phosphorylation of AMPK and raptor, an mTOR-interacting partner, which inhibited the mTOR-driven phosphorylation of P70S6K and eukaryotic translation initiation factor 4E binding protein. In the presence of compound C, deAND modulation of AMPK-mTOR signaling and inhibition of cell cycle regulator expression were reversed. Our results reveal that the anti-adipogenic effect of deAND is likely through inhibition of the PKA-CREB-C/EBPβ and AMPK/mTOR pathways, which leads to down-regulating C/EBPβ-driven lipogenic protein expression and halting MCE progression.