Genistein exerts antiadipogenic effects, but its target molecules remain unclear. Here, we delineated the molecular mechanism underlying the antiadipogenic effect of genistein. A pulldown assay using genistein-immobilized beads identified adenine nucleotide translocase-2 as a genistein-binding protein in adipocytes. Adenine nucleotide translocase-2 exchanges ADP/ATP through the mitochondrial inner membrane. Similar to the knockdown of adenine nucleotide translocase-2, genistein treatment decreased ADP uptake into the mitochondria and ATP synthesis. Genistein treatment and adenine nucleotide translocase-2 knockdown suppressed adipogenesis and increased phosphorylation of AMP-activated protein kinase. Adenine nucleotide translocase-2 knockdown reduced the transcriptional activity of CCAAT/enhancer-binding protein β, whereas AMP-activated protein kinase inhibition restored the suppression of adipogenesis by adenine nucleotide translocase-2 knockdown. These results indicate that genistein interacts directly with adenine nucleotide translocase-2 to suppress its function. The downregulation of adenine nucleotide translocase-2 reduces the transcriptional activity of CCAAT/enhancer-binding protein β via activation of AMP-activated protein kinase, which consequently represses adipogenesis.