Carbon fluxes in terrestrial ecosystems and their response to environmental change are a major source of uncertainty in the modern carbon cycle. The National Ecological Observatory Network (NEON) presents the opportunity to merge eddy covariance (EC)‐derived fluxes with CO₂ isotope ratio measurements to gain insights into carbon cycle processes. Collected continuously and consistently across >40 sites, NEON EC and isotope data facilitate novel integrative analyses. However, currently provisioned atmospheric isotope data are uncalibrated, greatly limiting ability to perform cross‐site analyses. Here, we present two approaches to calibrating NEON CO₂ isotope ratios, along with an R package to calibrate NEON data. We find that calibrating CO₂ isotopologues independently yields a lower δ¹³C bias (<0.05‰) and higher precision (<0.40‰) than directly correcting δ¹³C with linear regression (bias: <0.11‰, precision: 0.42‰), but with slightly higher error and lower precision in calibrated CO₂ mole fraction. The magnitude of the corrections to δ¹³C and CO₂ mole fractions vary substantially by site, underscoring the need for users to apply a consistent calibration framework to data in the NEON archive. Post‐calibration data sets show that site mean annual δ¹³C correlates negatively with precipitation, temperature, and aridity, but positively with elevation. Forested and agricultural ecosystems exhibit larger gradients in CO₂ and δ¹³C than other sites, particularly during the summer and at night. The overview and analysis tools developed here will facilitate cross‐site analysis using NEON data, provide a model for other continental‐scale observational networks, and enable new advances leveraging the isotope ratios of specific carbon fluxes.