We previously reported the lipase inhibitory activity of the n -BuOH fraction of Dioscorea opposita (DOB) and its isolates. This study sought to evaluate their anti-adipogenic activity in terms of their effects on the adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) as well as phosphorylated AMP-activated protein kinase (p-AMPK) and carnitine palmitoyl transferase-1 (CPT-1). DOB apparently attenuated 3T3-L1 adipocyte differentiation (33.6% decrease at 20 µg/mL). In addition, a marked decrease (90.4%) in the expression of PPARγ was observed in the DOB-treated 3T3-L1 cells. Four isolates from DOB: (4 E ,6 E )-1,7-bis(4-hydroxyphenyl)-4,6-heptadien-3-one ( 1 ), (3 R ,5 R )-1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanediol ( 2 ), batatasin I ( 3 ), and (1 E ,4 E ,6 E )-1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one ( 4 ), suppressed adipocyte differentiation by inhibiting PPARγ at 20 µM (85.9%, 68.6%, 76.2%, and 90.2% decrease, respectively) and C/EBPα (51.7%, 3.1%, 20.9%, and 59.8% decrease, respectively). Batatasin I was found to increase p-AMPK and CPT-1 at a concentration of 20 µM in 3T3-L1 adipocytes, resulting in inhibiting adipogenesis. Taken together, batatasin I might be responsible for the anti-adipogenic effect of DOB via inhibition of PPARγ and C/EBPα and activation of p-AMPK and CPT-1.