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.