Stream metabolism is a key biogeochemical process in river networks, synthesising the balance between gross primary production (GPP) and ecosystem respiration (ER). Globally, more rivers and streams are drying due to climate change and water abstraction for human uses and this can alter the organic carbon residence time, leading to decoupled ER and terrestrial organic matter supply. While the consequences of drying on CO2 emissions have been recently quantified, its effects on stream metabolism are still poorly studied. We addressed the short- and long-term effects of drying on stream metabolism by monitoring oxygen dynamics at 20 reaches across a drying river network, including perennial (PR) and non-perennial reaches (NPR) for one year. We also calculated several climatic, land use variables and characterized and local abiotic conditions, and biofilm and sediment communities at five sampling dates. ER was significantly higher in NPR than in PR reaches demonstrating in-situ the effects of drying on stream metabolism. When analyzing the long-term drivers of ER and GPP, we found a direct positive effect of drying on ER and a negative effect on GPP. Drying also altered microbial community composition, with algal communities from NPRs being different from those in PRs. In the short-term, the amount of C emitted during rewetting events was positively related to the duration of precedent non-flow period. Our results show that drying had an important effect on stream metabolism both in the short and long term, and supports the need of including NPRs in global estimates of stream metabolism.