C4 photosynthesis requires a combination of both biochemical and anatomical specialisation of leaf cells. Proliferation of plasmodesmata (PD) connections between bundle sheath (BS) and mesophyll (M) cells has been proposed as a key step in the evolution of C4 photosynthetic mechanism to support the high fluxes of photosynthetic metabolites between these cells. Lack of quantitative data has hampered further exploration and validation of this hypothesis. I developed a new methodology to quantify PD density in 3D leaf tissue. I used this technique to investigate PD density in 25 species of BEP and PACMAD grasses encompassing seven independent C4 lineages and all C4 biochemical subtypes (NADP-ME, NAD-ME, and PCK). I found that not only did C3 species have less PD than their C4 counterparts but within the C4 species there was also substantial variation of PD densities between the M and BS cells, where NAD-ME subtypes had the greatest value. This greater PD density in C4 leaves was a result of having larger pit fields (clusters of PD) and an increased number of PD per pit field area. I also showed that when C4 plants were subjected to different growth light environments or when the initial carbon fixation enzyme, PEPC, was downregulated, PD connections between the M and BS cells were altered. These results suggest that increased M-BS PD density is an essential anatomical feature of C4 photosynthesis and is responsive to changes in photosynthetic capacity in C4 plants.