Abstract: Field experiments aimed at the fertilizer 15N uptake, accumulation, distribution and use efficiency in flue-cured tobacco was conducted using 15N trace technique in yellow soil in Dafang county of Guizhou province. Two organic matter levels (19.2 and 40.70 g/kg) and two N application rates (105 and 82.5 kg/hm2) were involved. The results showed that only a small part of fertilizer 15N was absorbed in the first 3-5 weeks after transplanting (WAT). However, 15N accumulation increased rapidly since the fifth WAT and reached its maximum at the 13 WAT. The amount of 15N accumulation were 28.41 and 26.55 kg/hm2 for two organic matter level soil at harvest. The 15N uptake was increased with increasing N fertilizer applied. Ratio of N from 15N fertilizer to total N uptake decreased as the plant growth stages. It was ranged from 53.84% to 71.33% in the 3-5 WAT and 27.06% to 32.18% in the 17 WAT. The N use efficiency was 1.11% to 7.34% in 3-5 WAT and 28.69% to 29.75% in the 17 WAT. The 15N distribution at upper, middle, lower leaves and stem and flowers accounted for 35.08% to 35.26%, 25.87% to 26.19%, 17.92% to 18.25% and 22.73% to 24.49% to total 15N uptake respectively. Fertilizer 15N uptake was mainly cumulated in middle and upper leaves. The postponed fertilizer15N absorption, unsynchronized supplication of soil mineralized N at late growth stage, and higher 15N accumulation in upper and middle leaves of tobacco were the key problems of nutrient management of tobacco to deal with in yellow soil.