The photosynthetic performance of C4 plants is generally inferior to that of C3 species at low temperatures, but the reasons for this are unclear. The present study investigated the hypothesis that the capacity of Rubisco, which largely reflects Rubisco content, limits C4 photosynthesis at suboptimal temperatures. Photosynthetic gas exchange, chlorophyll a fluorescence, and the in vitro activity of Rubisco between 5 and 35 °C were measured to examine the nature of the low-temperature photosynthetic performance of the co-occurring high latitude grasses, Muhlenbergia glomerata (C4) and Calamogrostis canadensis (C3). Plants were grown under cool (14/10 °C) and warm (26/22 °C) temperature regimes to examine whether acclimation to cool temperature alters patterns of photosynthetic limitation. Low-temperature acclimation reduced photosynthetic rates in both species. The catalytic site concentration of Rubisco was approximately 5.0 and 20 µmol m−2 in M. glomerata and C. canadensis, respectively, regardless of growth temperature. In both species, in vivo electron transport rates below the thermal optimum exceeded what was necessary to support photosynthesis. In warm-grown C. canadensis, the photosynthesis rate below 15 °C was unaffected by a 90% reduction in O2 content, indicating photosynthetic capacity was limited by the capacity of Pi-regeneration. By contrast, the rate of photosynthesis in C. canadensis plants grown at the cooler temperatures was stimulated 20–30% by O2 reduction, indicating the Pi-regeneration limitation was removed during low-temperature acclimation. In M. glomerata, in vitro Rubisco activity and gross CO2 assimilation rate were equivalent below 25 °C, indicating that the capacity of the enzyme is a major rate limiting step during C4 photosynthesis at cool temperatures.