Assessing the origin, transformation and transport of terrestrially derived carbon in river systems is critical to regional and global carbon cycles, particularly in carbonate terrains, which represent the largest carbon reservoir on the earth’s surface. For this reason, we evaluated sources, cycling, and fluxes of dissolved organic and inorganic carbon (DOC and DIC) and riverine CO2 degassing to the atmosphere in the Santa Fe River in north-central Florida, a sub-tropical river that flows across two distinct hydrogeological settings of a region dominated by carbonate karst. One setting occurs in the upper river catchment, where the carbonate Floridan aquifer is confined by the siliciclastic Hawthorn Group, while the other setting occurs in the lower catchment where the river flows across the unconfined Floridan aquifer. The upper catchment is characterized by DOC-rich and DIC-poor water and the DIC has more variable and lower δ13C values compared to the lower catchment. The river in the upper catchment degasses more CO2 to the atmosphere (1156 g C m−2 yr−1) than in the lower catchment (402 g C m−2 yr−1) because soil respired carbon and organic matter decomposition increase dissolved CO2 concentration, much of which is consumed during carbonate dissolution reactions in the lower catchment. The CO2 flux from the water surface to the atmosphere during a flood event is three times greater than during base flow, suggesting that excess precipitation flushes soil organic carbon to the river through interflow and enhances the loss of terrestrial carbon via river water to the atmosphere. Our values of CO2 fluxes to the atmosphere lie within the range of fluxes from the world’s rivers, but fluxes from the carbonate dominated region are at the low end, while fluxes from the siliciclastic region are at the high end. These results indicate that catchment lithologies, particularly whether carbonate or siliciclastic, as well as flow, are critical to carbon budgets in rivers and thus are linked to the global carbon cycle.