We assessed the effects of landscape change on the climate regulation ecosystem service in a mountain river basin of Portugal, through the quantification, valuation and mapping of carbon sequestration and storage. The analyses were based on land use and land cover (LULC) changes that took place between 1990 and 2006 and on expected changes defined by three LULC change scenarios for 2020. We used the Integrated Valuation of Ecosystem Services and Tradeoffs model for scenario building and carbon assessment and valuation, and several modelling tools to assess past, current and future carbon in four different pools. Soil organic carbon data was obtained through an extensive sampling scheme across the entire study area. Recent (1990–2006) and expected landscape changes (2006–2020) affected considerably carbon sequestration and storage. Observed landscape changes generally promoted carbon sequestration and storage, and had a positive effect on the climate regulation ecosystem service, both biophysically and economically. Expected LULC changes further extend the capability of the landscape to increase carbon sequestration and storage in the near future. The carbon sequestered and stored in vegetation and soil contributes to avoid socio-economic damages from climate change, while increasing the economic value of particular LULC classes and the whole landscape. These results are essential to inform land planning, especially on how, where and when changes in landscapes may affect the provision of the climate regulation ecosystem service.