Abstract: To investigate the interaction effects of different planting density and nitrogen (N) application amount on tobacco leaf development and tissue structures, a split-plot field experiment with three different planting densities (main district) and two different N application amounts (deputy district) was carried out using the cultivar NC102. Through paraffin section method, the tissue structure of middle leaves with different leaf ages (10, 20, 40 and 73 d) were observed. The results showed that leaf thickness, palisade tissue thickness, spongy tissue thickness and leaf area increased with leaf aging, but palisade cell density decreased. Leaf thickness, palisade tissue thickness, spongy tissue thickness, leaf area and specific leaf weight increased but palisade cell density decreased with the decrease of planting density and increased N application rate. The action time of N application amount on palisade cell density was earlier than that of planting density, while the timing of effects of these two treatments was the opposite for palisade tissue thickness. There was a positive interaction between low planting density and high N application amount on leaf thickness, palisade tissue thickness, ratio of palisade tissue thickness and spongy tissue thickness, leaf area and specific leaf weight when leaf age was 73 d. The increase of N application amount showed more significant impact under lower planting densities. Adopting appropriate planting density and N application amount and taking advantage of their interaction could improve tissue structures of middle leaves and ultimately improve the quality of tobacco under proper yield.