This study quantifies small-scale carbon dioxide (CO₂) efflux and estimates annual CO₂ emission from a headwater stream at the Konza Prairie Long-Term Ecological Research Site and Biological Station (Konza), in a terrain of horizontal, alternating limestones and shales. We characterized the CO₂ efflux and stable carbon isotopes (δ¹³C-CO₂) at point sources of groundwater discharge from small-scale karst features, identified by temperature, and downstream of those point sources, as well as in stream reaches without identifiable point sources. CO₂ effluxes ranged from 2.2 to 214 g CO₂ m^-2 day^-1 (mean was 20.9± 41.4 g CO₂ m^-2 day^-1). Downstream of point groundwater discharge sources, CO₂ efflux decreased, over 2 meters, to 3% to 40% of the point-source flux, while δ¹³C-CO₂ increased, ranging from -9.8 ‰ to -23.2 ‰ V-PDB (mean was -14.9 ‰ ± 4.2 ‰ V-PDB). The δ¹³C-CO₂ increase was not strictly proportional to the CO₂ flux but related to the origin of vadose-zone CO₂ (C3 versus C4 vegetation). Over the study period, ~7.0 metric tons of CO₂ were emitted from the 1.1-km-long stream, comparable to other headwater streams. The high spatial and temporal variability of CO₂ efflux from this headwater stream informs those doing similar measurements and those working on upscaling stream data, that local variability should be assessed to make the best estimate of the impact of headwater stream CO₂ efflux on the global carbon cycle.