The effects of juvenile hormone III (JH III) on the premolting period and mortality of nymphal Haemaphysalis longicornis, and 2,6-dichlorophenol (2,6-DCP) synthesis in its female were determined. Nymphs were treated topically with 1, 5 and 10 μg/tick JH III on day 2 (2-PE), day 7 (7-PE), and day 10 (10-PE) post engorgement. The premolting periods of nymphs were prolonged by all doses in nymphs of the 2-PE group, but were normal in those of the 7-PE and 10-PE groups. With the increase of doses, the mortality rose in all groups. Inoculation of JH III on newly molted females enhanced the level of 2,6-DCP synthesis. Our results suggest that JH III is involved in the regulation of nymphal development and synthesis of 2,6-DCP in females of this tick species.