A fundamental limitation to photosynthetic carbon fixation is the availability of CO2. The significant contribution of aquaporins to increased CO2 diffusion from intercellular airspace to the mesophyll cytosol (mesophyll conductance) has been demonstrated in C3 plants. However, to date nothing is known about the role of CO2 permeable aquaporins in the highly efficient and specialised C4 photosynthetic pathway. In the C4 pathway, CO2 is initially fixed as bicarbonate by phosphoenolpyruvate carboxylase in the mesophyll cytosol, rather than by Rubisco in the mesophyll chloroplast, as is the case in C3 plants. This raises the question of whether CO2 permeable aquaporins are required in C4 plants using the C4 pathway. We have examined the role of aquaporins of the model C4 monocot species Setaria viridis (Green Foxtail millet) using yeast as a heterologous expression system. All nine Setaria PIPs (plasma membrane intrinsic proteins) were constitutively expressed in yeast and plasma membrane localisation was confirmed. CO2 permeability of the plasma membrane was measured using two techniques: stopped flow spectrophotometry and membrane inlet mass spectrometry. We identified one candidate, SiPIP2.6, which showed significant CO2 permeability (PCO2 = 1.9 × 10–4 m s–1) compared to the control (PCO2 = 0.8 × 10–4 m s–1). The importance of CO2 permeable aquaporins in planta is currently being investigated in the C4 plant Setaria.