Abstract: In order to investigate the response of root growth, C and N accumulation of flue-cured tobacco seedlings to different nitrogen forms, hydroponic experiments were conducted by supplying equal amount of N in three different ratios of NH₄⁺:NO₃^- (100:0, as 100-0AN; 50:50, as 50-50AN; 0:100, as 0-100AN) to seedlings of flue-cured tobacco cultivar NC55. Plant dry biomass, root morphology, C and N content, C and N accumulation were assayed during the cultivation. The results showed that shoot and total biomass showed 0-100AN> 50-50AN> 100-0AN at all treating times. On 7 and 14 d, root biomass, root total length, root total volume, root total surface area, C and N accumulation of root, stem, leaf and total nitrogen accumulation showed 0-100AN> 50-50AN> 100-0AN. On 21 d, the same parameters showed 50-50AN> 0-100AN> 100-0AN. Ammonium as sole nitrogen source inhibited flue-cured tobacco growth. Nitrate is beneficial to shoot growth and carbon accumulation, it however inhibits root growth as sole nitrogen source for prolonged time. Optimum nitrogen form for root development, C and N accumulation is the mixture of ammonium and nitrate.