We examined carbon cycling in the Mississippi River using stable isotopes of inorganic carbon and dissolved oxygen. Eighteen sites were sampled along the river and its tributaries over 1 year. We estimate using a conservative approach that the flux of CO2 to the atmosphere (1 × 1013 g C yr−1) approximately equaled the flux of alkalinity to the Gulf of Mexico (9.7 × 1012 g C yr−1) and greatly exceeded the flux of dissolved organic carbon (1.5 × 1012 g C yr−1). Though only a first‐order estimate, our work shows that the atmospheric flux of CO2 is significant and should not be ignored when examining the carbon budget of the Mississippi River. As expected, because of the large area covered by the Mississippi watershed, the isotopic composition of dissolved inorganic carbon, δ13CDIC, varied widely. In the Ohio and upper and lower Mississippi basins, δ13CDIC indicates that the source of inorganic carbon in the rivers is primarily from carbonate dissolution by soil CO2. Dissolved inorganic carbon in the Missouri River was enriched in 13C, and the isotopic composition of dissolved oxygen in this river suggests that this results from an excess of aquatic photosynthesis over respiration.