Abstract: In order to provide theoretical guidance for high quality tobacco breeding, analysis of contribution from nicotine transport related factors to nicotine accumulation was conducted. Seven flue-cured tobacco germplasms with different nicotine contents were planted in hydroponics devices and were treated by topping. The nicotine content, and expression levels of 2 key nicotine biosynthetic genes (NtPMT, NtQPT) and 3 transporters genes (NtNUP1, NtMATE1/2, NtJAT1/2) and a vesicular traffic regulator gene (NtGEF) were detected and analyzed. Correlation analysis, principal component analysis and related network analysis were conducted. The results showed that:(1) expression of nicotinic biosynthetic genes (NtPMT and NtQPT) and nicotine transporter genes (NtNUP1 and NtMATE1/2) was promoted by each other; (2) nicotine transporter genes that play dominant roles in leaves and roots were different. NtJAT1 was the dominant transporter in middle leaves; NtNUP1, NtJAT1 and NtMATE1/2 genes were the main genes of nicotine transportation in the root, with their transporting efficiency decreased in the above listed order; (3) the vesicular traffic regulator gene (NtGEF) also indirectly affected the accumulation of nicotine in middle leaves. Therefore, when seeking directional improvement for nicotine content, breeders should consider the influence of nicotine synthesis genes, transporter genes and vesicle transport regulatory factors on nicotine accumulation.