Vegetated ditches and wetlands are important sites for nutrient removal in agricultural catchments. About half of the influx of inorganic nitrogen can be removed from these ecosystems by denitrification. Previous studies have shown that denitrification in aquatic ecosystems is strongly temperature dependent, resulting from temperature-dependent oxygen availability. Here, we study short-term temperature effects on sediment oxygen demand (SOD) and the maximum depth of oxygen penetration into the sediment (Z), in relation to overall denitrification rates. We set up sixteen wetland microcosms at four different temperatures (11–25 °C), in which we determined SOD and Z from sediment oxygen microprofiles. Denitrification rates were measured using 15N-labeling, analysed by membrane inlet mass spectrometry. Temperature strongly affected sediment oxygen dynamics. SOD exponentially rose with temperature, ranging from 0.37 to 1.53 g m−2 d−1 (Q10 = 2.4). Correspondingly, warming led to shallower oxygen penetration into the sediment, ranging from 4.12 to 2.08 mm. Denitrification rates increased with warming (Q10 = 2.6), ranging from 8.4 to 86 μmol N m−2 h−1. The results of this short-term experiment confirm the potential increase of denitrification with rising temperature, promoted by lower oxygen availability in the top layer of the sediment, which supports the understanding of denitrification variability in freshwaters.