Combined application of TPx methods, H2‐O2 titration, UV–vis DRS, TGA‐DSC‐MS, TEM, XRD, N2 adsorption at −196 oC allowed proving the OH/Ag molar ratio as the key parameter defining the catalytic properties of silica-supported silver (Ag/SiO2) in low‐temperature CO oxidation. A new insight into the formation of active species on the catalyst surface is presented. In this study, Ag/SiO2 catalysts with Ag loading of 5 and 8 wt.% were prepared by incipient wetness impregnation method on the basis of commercial silicas preliminary calcined at 500, 700 and 900 °C. Detailed characterization of catalysts by physicochemical methods revealed that molar ratio between the concentration of surface OH groups (normalized to support mass) and silver amount in the prepared catalysts (OH/Ag ratio) affects the silver dispersion, structure of silver nanoparticles (NPs) and their catalytic properties. Only at optimal value of OH/Ag ratio the silver NPs are stated to possess both high dispersion and defective multidomain structure (that consists of several nanodomains) providing the adsorption of weakly bound oxygen species responsible for high catalytic activity in CO oxidation at low‐temperature. Additionally, the co-existence of two types of active sites reacting with CO at room temperature with and without formation of adsorbed carbonate species is discussed. The first type of active sites was catalytically active in low-temperature CO oxidation with CO2 release at room temperature (RT). The second type may retain surface carbonate species up to ∼40–50 °C. The balance between these species shifts towards the first type for the active catalyst.