The inertness of graphene toward reaction with ambient molecules is essential for realizing durable devices with stable performance. Many device applications require graphene to contact with substrates, but whose impact on the chemical property of graphene has been largely overlooked. Here, we combine comprehensive first-principles analyses with experiments to show that graphene oxidation is highly sensitive to substrates. Graphene remains inert on SiO2 and hexagonal boron nitride but becomes increasingly weak against oxidation on metal substrates because of enhanced charge transfer and chemical interaction between them. In particular, Ni and Co substrates lead to spontaneous oxidation of graphene, while a Cu substrate maximally promotes the oxygen diffusion on graphene, with an estimated diffusivity 13 orders of magnitude higher than that on freestanding graphene. Bilayer graphene is revealed to have high oxidation resistance independent of substrate and thus is a better choice for high-performance nanoelectronics. Our findings should be extendable to a wide spectrum of chemical functionalizations of two-dimensional materials mediated by substrates.