The effect of acid treatment on manganese oxide samples has been investigated for catalytic oxidation of toluene. The acid treatment had no obvious influence on the textural properties of catalysts, but could remarkably improve the catalytic performance of catalysts, especially the catalysts acid-treated with low acid concentration. The acid-treated catalysts exhibited the relatively high amount of surface Mn4+ along with the probable formation of vacancy defects via the disproportionation of Mn3+ by acid leaching. The structural defects could further contribute to the good surficial oxygen mobility and the low-temperature reducibility, which is indispensable for the significant enhancement of toluene oxidation. As a result, the specific toluene reaction rate increased and the activation energy decreased with the increase of acid concentration. In situ DRIFTS results indicated that the intermediates such as alkoxide, benzaldehyde, benzoate, and maleic anhydride species were produced during the toluene oxidation process.