In this study, a series of chlorine-modified molybdenum catalysts supported on silica and titania (Si:Ti) mixed oxides were prepared by a modified sol–gel technique. The catalytic performance of the catalysts was tested for the oxidative dehydrogenation (ODH) of propane. Catalysts were characterized by in situ or controlled-atmosphere measurements with XPS, LRS, ESR, and DRIFTS techniques to elucidate the adsorption and transformation of propane/propylene and the reduction of surface molybdena species over the Si:Ti support. In addition, isotopic labeling studies have been performed to investigate the oxygen mobility and oxygen exchange characteristics and reaction pathways involving propane ODH. These characterization results are correlated with the catalytic reaction performance to achieve a better understanding of the catalytic behavior of chlorine-modified Mo/Si:Ti catalysts.