The aim of the present study was to investigate effects of the ethyl acetate fraction of an ethanol extract of Coix lachryma-jobi (ECLJ) on glucose uptake and adipocyte differentiation in 3T3-L1 cells. ECLJ phosphorylated AMP-activated protein kinase (AMPK) and its downstream substrate acetyl-coenzymeA carboxylase in 3T3-L1 cells in a time- and dose-dependent manner. Moreover, we discovered that compound C inhibits ECLJ-stimulated ACC phosphorylation. In addition, ECLJ exhibited a dose-dependent stimulation of glucose uptake in 3T3-L1 cells, and this increase was obviously attenuated by compound C. ECLJ also caused a decrease in the expression levels of adipogenesis factors such as fatty acid synthase, sterol-regulatory-element-binding protein-1c, peroxisome proliferator-activated receptor γ, and CAATT/enhancer binding protein α in a dose-dependent manner. Differentiation was examined by Oil red O staining activity after ECLJ treatment for 6 days. ECLJ decreased mean droplet size. These results suggest a possible role for AMPK in the process of adipose differentiation and that ECLJ targeted for adipocyte functions could be effective in improving the symptoms of metabolic syndrome.