The acidic properties of alumina-supported molybdenum oxide (MoO3/Al2O3) calcined at high temperatures, with MoO3 loadings of 5-30 wt%, were investigated using acid-catalyzed reactions (benzylation of anisole and isomerization of alpha-pinene), and Fourier-transformed infrared spectroscopy (FT-IR). The structure of MoO3 on Al2O3 was characterized by X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectra (XPS), and X-ray absorption fine structure (XAFS) analysis. The correlation between acidic property and structure is discussed. Bronsted acid sites, where acid-catalyzed reactions take place, are generated on MoO3/Al2O3 by calcination at high temperatures. 11 wt% MoO3/Al2O3 calcined at 1073 K exhibited the highest activity, and the largest numbers of Bronsted acid sites were generated. XPS and Mo K-edge XAFS revealed that molybdenum oxide monolayer domains with distorted MoO6 units and small MoO3 clusters were formed and molybdenum oxide monolayer domains were stabilized on alumina below 11 wt% of MoO3 loading. Bronsted acid sites are probably generated at boundaries between molybdenum oxide monolayer domains and/or small MoO3 clusters. When the MoO3 loading was sufficient to form two-dimensional molybdenum oxide overlayers (>11 wt%), some of the Bronsted acid sites on MoO3/Al2O3 was covered with Al2Mo3O12 having no Bronsted acid site, resulting in a lowering of the catalytic activity. (C) 2013 Elsevier B.V. All rights reserved.