Solution pot experiments were conducted to study the effects of 2, 4-D on the activities of nitrate reductase (NR), invertase (InV), ornithine decarboxylase (ODC), putrescine N-methyl transferase (PMT), and N-methyl-putrescine oxidase (MPO), and on the contents of nicotine and potassium in flue-cured tobacco. The results showed that, by spraying 2, 4-D on tobacco leaves after topping, leaf InV activity decreased, NR activity increased, carbon-nitrogen metabolism was better balanced, root activity decreased, three enzyme activities in nicotine synthesis of tobacco roots were constrained, nicotine accumulation decreased, and potassium content in tobacco leaves increased. The optimum 2,4-D concentrations were 20 mg/kg and 30 mg/kg for decreasing nicotine content and increasing potassium content, respectively.