Stable isotopes provide insight into ecosystem carbon cycling, plant physiological processes, atmospheric boundary-layer dynamics, and are useful for the integration of processes over multiple scales. Of particular interest is the carbon isotope content (δ¹³C) of nocturnal ecosystem-respired CO₂ (δ_R). Recent advances in technology have made it possible to continuously examine the variation in δ_R within a forest canopy over relatively long time-scales (months–years). We used tunable diode laser spectroscopy to examine δ_R at within- and below-canopy spatial locations in a Colorado subalpine forest (the Niwot Ridge AmeriFlux site). We found a systematic pattern of increased δ_R within the forest canopy (δ_R-c) compared to that near the ground (δ_R-g). Values of δ_R-c were weakly correlated with the previous day's mean maximum daytime vapor pressure deficit (VPD). Conversely, there was a negative but still weak correlation between δ_R-g and time-lagged (0–5 days) daily mean soil moisture. The topography and presence of sustained nightly drainage flows at the Niwot Ridge forest site suggests that, on nights with stable atmospheric conditions, there is little mixing of air near the ground with that in the canopy. Atmospheric stability was assessed using thresholds of friction velocity, stability above the canopy, and bulk Richardson number within the canopy. When we selectively calculated δ_R-g and δ_R-c by removing time periods when ground and canopy air were well mixed, we found stronger correlations between δ_R-c and VPD, and δ_R-g and soil moisture. This suggests that there may be fundamental differences in the environmental controls on δ_R at sub-canopy spatial scales. These results may help explain the wide variance observed in the correlation of δ_R with different environmental parameters in other studies.