Abstract: To study the effects of exogenous growth regulator Z5 (IAA+NAA+SA) on the biosynthesis of tobacco-specific N-nitrosamines (TSNAs), tobacco leaves were sprayed with Z5 (water as the control) by pot experiments in Jianshi County and Enshi City in Hubei Province. The dynamic variations of nitrate and nitrite contents, the activities of NR (nitrate reductase) and key enzymes related to nicotine synthesis, and the expressions of related genes were analyzed. The TSNA contents and routine chemical components in cured leaves were determined. The results showed that TSNA contents in leaves sprayed with Z5 after toping significantly reduced by 20.1% and 12.4% in upper and middle leaves, respectively. However, the contents of chemical components in leaves were in an appropriate range. The mechanism of Z5 to reduce TSNA contents were two:1) The expression of NR which controlled NR activity was down-regulated by 55.0% on the 1st day after topping. The conversion of nitrate to nitrite decreased due to the inhibition of NR activity, then the accumulation of nitrite reduced. 2) The expressions of key genes (ADC, QPRT, ODC, and PMT) related to nicotine biosynthesis were down-regulated by 82.1%, 69.8%, 91.8% and 76.0% on the 1st day after topping respectively, which resulted in the reduction of key enzymes' activities in nicotine synthesis. Finally the synthesis of nicotine was inhibited. Nicotine contents in upper and middle leaves decreased by 23.5% and 22.9%, respectively.