Abstract Perennial species with the C4 pathway hold promise for biomass-based energy sources. We have explored the extent that CO2 uptake of such species may be limited by light in a temperate climate. One energetic cost of the C4 pathway is the leakiness (ϕ) of bundle sheath tissues, whereby a variable proportion of the CO2, concentrated in bundle sheath cells, retrodiffuses back to the mesophyll. In this study, we scale ϕ from leaf to canopy level of a Miscanthus crop (Miscanthus × giganteus hybrid) under field conditions and model the likely limitations to CO2 fixation. At the leaf level, measurements of photosynthesis coupled to online carbon isotope discrimination showed that leaves within a 3.3-m canopy (leaf area index = 8.3) show a progressive increase in both carbon isotope discrimination and ϕ as light decreases. A similar increase was observed at the ecosystem scale when we used eddy covariance net ecosystem CO2 fluxes, together with isotopic profiles, to partition photosynthetic and respiratory isotopic flux densities (isofluxes) and derive canopy carbon isotope discrimination as an integrated proxy for ϕ at the canopy level. Modeled values of canopy CO2 fixation using leaf-level measurements of ϕ suggest that around 32% of potential photosynthetic carbon gain is lost due to light limitation, whereas using ϕ determined independently from isofluxes at the canopy level the reduction in canopy CO2 uptake is estimated at 14%. Based on these results, we identify ϕ as an important limitation to CO2 uptake of crops with the C4 pathway.