Birnessite was synthesized by a simple hydrothermal method and used for low-temperature selective catalytic reduction of NO with NH3 (NH3-SCR). Physicochemical characterizations showed that the exposure of surface oxygen vacancies on birnessite was affected by heat treatment through the removal of water molecules. The sample heat-treated at a temperature of 150 degrees C, having the most exposed surface oxygen vacancies, exhibited excellent catalytic activity with 100% NO removal rate at 99 degrees C. In addition, density functional theory (DFT) calculations were combined with TPD techniques and in situ Fourier transform infrared (FTIR) to disclose the mechanism of NH3-SCR over birnessite. The results indicated that surface oxygen vacancies played a vital role in adsorption and oxidiation of reactant molecules and participated in the formation of acidic sites. This study shows that regulation of the exposure of surface oxygen vacancies of birnessite significantly improves its low-temperature NH3-SCR catalytic activity.