Although various Pd/C catalysts have been applied in the direct synthesis of H2O2, unsatisfactory H2O2 yields have been achieved. We systematically investigated the effect of heat treatment on the physicochemical properties of Pd/C catalyst, and thereby on the catalytic performance in the direct synthesis of H2O2. Pd/C catalysts prepared by the incipient wetness method were subjected to different heat treatments and applied in H2O2 synthesis. The calcination temperature was found to have a key role in determining the Pd nanoparticle (NP) size; calcination at 523 K yielded highly oxidized and small Pd NPs corresponding to the sub-nano domain (1.4-2.5 nm). This Pd/C catalyst is superior not only in promoting H2O2 formation, but also in suppressing the subsequent unfavorable H2O2 decomposition and hydrogenation, which explains its excellent H2O2 productivity (as high as 4,443mmol H2O2/g Pd·h) and selectivity (94.5%). On the other hand, the reaction performance of the Pd/C catalysts calcined at a higher temperature (673 K) or reduced under hydrogen was sharply reduced owing to the formation of larger Pd NPs or the enhancement of the metallic nature of Pd, respectively. The amount of residual Cl ion on Pd/C catalyst after heat treatment also had an impact on the catalytic activity as it affected the pH of reaction solution. These results clearly demonstrate that an efficient Pd/C catalyst can be realized by fine tuning the conditions of heat treatment during catalyst preparation.