Mycelial extracts have a wide range of biological activities that modulate functions of mammalian cells. In this report, we sought anti-adipogenic mycelia using 3T3-L1 cells and found that the extract of Cordyceps militaris (C. militaris) exclusively suppressed differentiation of 3T3-L1 preadipocytes into mature adipocytes without affecting cell viability. This inhibitory effect was dose-dependent, reversible, and associated with; 1) decreases in lipid accumulation, 2) blunted induction of adipocyte markers including adiponectin, peroxisome proliferator-activated receptor and CCAAT/enhancer binding protein , and 3) sustained expression of a preadipocyte marker monocyte chemoattractant protein 1. C. militaris also significantly decreased accumulation of lipid and hypertrophy in mature adipocytes and preserved their response to insulin (phosphorylation of Akt) during prolonged culture. Subsequent experiments revealed that C. militaris has the potential to activate the aryl hydrocarbon receptor (AhR). In 3T3-L1 cells, treatment with AhR agonists including benzo[a]pyrene and 3-methylcholanthrene reproduced the anti-adipogenic effect of C. militaris. Furthermore, dominant-negative inhibition of AhR abrogated the suppressive effect of C. militaris on adipocyte differentiation. These results suggested that C. militaris has the potential for interfering with adipocyte differentiation through activation of AhR.