The mechanism of photosynthesis in C4 crops depends on the archetypal Kranz-anatomy. To examine how the leaf anatomy, as altered by nitrogen supply and leaf age, affects the bundle sheath conductance (gbs), maize (Zea mays L.) plants were grown under three contrasting nitrogen levels. Combined gas exchange and chlorophyll fluorescence measurements were done on fully grown leaves at two leaf ages. The measured data were analysed using a biochemical model of C4 photosynthesis to estimate gbs. The leaf microstructure and ultrastructure were quantified using images obtained from micro-computed tomography and microscopy. There was a strong positive correlation between gbs and leaf nitrogen content (LNC) while old leaves had lower gbs than young leaves. Leaf thickness, bundle sheath cell wall thickness and surface area of bundle sheath cells per unit leaf area (Sb) correlated well with gbs although they were not significantly affected by LNC. As a result, the increase of gbs with LNC was little explained by the alteration of leaf anatomy. In contrast, the combined effect of LNC and leaf age on Sb was responsible for differences in gbs between young leaves and old leaves. Future investigations should consider changes at the level of plasmodesmata and membranes along the CO2 leakage pathway to unravel LNC and age effects further.