Abstract Carbon isotope composition of dissolved inorganic carbon (DIC) was studied at springs, lakes and tributaries of the Plitvice Lakes. The Plitvice Lakes are a unique and complex karst lakes system consisting of 16 flow-through lakes connected by waterfalls and streams and characterized by intense calcium carbonate precipitation in the form of tufa and lake sediments. Two main springs have different 14 C concentrations due to different geology and their variations were controlled by flow rates. Further downstream, at lakes to the Korana River that outflows from the lakes, the 14 C activity and δ 13 C consistently increased down the flow. Carbon isotope composition from 2010 to 2015 was compared with the values measured 30 years earlier. The variation in δ 13 C DIC was accounted for seasonal change, while 14 C activity of surface water DIC decreased 7–12% during the 30 years. Using a semi-empiric model, it is calculated that the downstream increase is controlled by the exchange of DIC and atmospheric CO 2 carbon and by introduction of decomposed biogenic carbon from the top soil organic matter of the lakes surrounding area in almost equal proportions.