Abstract: In order to explore the effect of soil water content on the infection of tobacco cyst nematode (Heterodera glycines) and the possible mechanism, pot experiments with three water treatments were carried out from June to September of 2014, the number of different-staged nematodes in soil and root and the nicotine content of tobacco leaf and root were determined. The results showed that, under normal water treatment, cyst nematode obviously occurred 3 generations, with a high reproduction factor (Rf=Pf/Pi) of 2.7, and the nicotine content in tobacco roots was lower than 1.00%. In the overwatering treatment in August, the third generation of cyst nematode barely occurred and was retarded, with the Rf of 0.5, and nicotine content in tobacco roots reached the maximum (1.92%) 15 d after the overwatering treatment. In the overwatering treatment in July and August, the third generation of cyst nematode barely occurred and both the second generation and the third one were retarded, with a small Rf of 0.1, and the maximum nicotine content (3.22%) in tobacco roots was reached 35 d after the overwatering treatment. The nicotine content of tobacco leaf showed an overall rising trend under normal water treatment and decreased in both overwatering treatments. Overwatering obviously inhibited the infection of tobacco cyst nematode, which was somehow correlated with the accumulation of nicotine in tobacco roots.