The activity of CaNaY zeolites in selective oxidation of propane with increasing Ca2+ content at room temperature was studied with infrared spectroscopy and ammonia temperature-programmed desorption. Increasing the Ca2+ exchange level in CaNaY zeolites resulted in a variety of altered properties: the amount of Bronsted and Lewis acid sites increased, the adsorption of propane increased, the initial acetone formation rate increased, and the desorption of acetone retarded. Moreover, all of these properties showed a sudden change with Ca2+ content above the 50% Na+ exchange level, which can be fully attributed to the location of Ca2+ in the Y-zeolite framework. It is convincingly shown that only Ca2+ ions located in the supercage contribute to the formation of Bronsted and Lewis acidity as well as propane oxidation activity at room temperature.