Land surface models are useful tools to quantify contemporary and future climate impact on terrestrial carbon cycle processes, provided they can be appropriately constrained and tested with observations. Stable carbon isotopes of CO2 offer the potential to improve model representation of the coupled carbon and water cycles because they are strongly influenced by stomatal function. Recently, a representation of stable carbon isotope discrimination was incorporated into the Community Land Model component of the Community Earth System Model. Here, we tested the model's capability to simulate whole-forest isotope discrimination in a subalpine conifer forest at Niwot Ridge, Colorado, USA. We distinguished between isotopic behavior in response to a decrease of δ13C within atmospheric CO2 (Suess effect) vs. photosynthetic discrimination (Δcanopy), by creating a site-customized atmospheric CO2 and δ13C of CO2 time series. We implemented a seasonally-varying Vcmax model calibration that best matched site observations of net CO2 carbon exchange, latent heat exchange and biomass. The model accurately simulated observed δ13C of needle and stem tissue, but underestimated the δ13C of bulk soil carbon by 1–2 ‰. The model overestimated the multi-year (2006–2012) average Δcanopy relative to prior data-based estimates by 5–6 ‰. The amplitude of the average seasonal cycle of Δcanopy (i.e. higher in spring/fall as compared to summer) was correctly modeled but only with an alternative nitrogen limitation formulation for the model. The model attributed most of the seasonal variation in discrimination to the net assimilation rate (An), whereas inter-annual variation in simulated Δcanopy during the summer months was driven by stomatal response to vapor pressure deficit. Soil moisture did not influence modeled Δcanopy. The model simulated a 10 % increase in both photosynthetic discrimination and water use efficiency (WUE) since 1850 as a result of CO2 fertilization, forced by constant climate conditions. This increasing trend in discrimination is counter to well-established relationships between discrimination and WUE. The isotope observations used here to constrain CLM suggest 1) the model overestimated stomatal conductance and 2) the default CLM approach to representing nitrogen limitation (post-photosynthetic limitation) was not capable of reproducing observed trends in discrimination. These findings demonstrate that isotope observations can provide important information related to stomatal function driven by environmental stress from VPD and nitrogen limitation.