Methane (CH₄) emissions from river systems contribute to the global greenhouse gas budget, but their contribution remains poorly constrained. Although proxies such as temperature, electron acceptor (EA) availability, and microbial communities are controlling factors, the role of the sediment characteristics (sediment permeability and organic carbon (OC) content) on anaerobic oxidation of methane (AOM) coupled with nitrate (NO₃^-)/nitrite (NO₂^-) is not well unterstood. Here, we investigated gravel-dominated, high-permeability sediments, where efficient advective transport promotes the deep penetration of terminal EAs such as oxygen (O₂), NO₃^- and sulfate (SO₄^2-) and inhibits microbial methane production. Conversely, hyporheic zone (HZ) sediment with fine-grained sediment was characterized by lower permeability, which restricts solute transport and facilitates diffusion-dominated processes and the development of anaerobic zones. Under these conditions, the presence of microbial available OC may support biological methane formation. Analyses of microbial communities of two profiles further indicate that also the distribution of methanogenic and methane-oxidizing taxa is closely linked to sediment permeability-controlled geochemical zonation. The 1D reactive transport modeling suggests that AOM with NO₃^- and NO₂^- as dominant EAs is selected for as a microbial process in the lower permeable sediments. Sediment permeability, therefore, regulated EA availability and, together with OC availability, shapes geochemical zonation, microbial community structure and the mechanism of AOM in the HZ. Therefore, sediment characteristics are shown here to strongly influence transport-reaction coupling, thereby regulating AOM and ultimately methane emissions from the HZ.