Abstract: In order to study the effects of nitrogen application rate and flowering date on the formation of fiber fineness, maturity and micronaire, two field experiments were carried out in Nanjing (118o50E, 32o02N) and Xuzhou (11711E, 3415N), standing for the different ecological conditions in the middle lower reaches of Yangtze River Valley and the Yellow River Valley in China. Three N levels were set up as N 0, 240 and 480 kg/ha, standing for low, medium and high nitrogen level respectively, and the two cultivars were selected as Kemian 1 and NuCOTN 33B with different fiber quality indices. The result showed that: (1) Nitrogen application rate significantly influence cotton fiber fineness, maturity and micronaire, but the responses of these three indices to N were different. The highest fiber fineness and micronaire was found in N 0 kg/ha before 10-Aug (daily mean temperature was higher than 23.3 oC), while in 25-Aug (daily mean temperature was between 20.8 and 23.3 oC), it was found highest in the treatment of N 240 kg/ha and in 10-Sep (daily mean temperature was lower than 20.8 oC) under the treatment of N 480 kg/ha. (2) The variation of final cotton fiber fineness, maturity and micronaire under different nitrogen application rates was smaller than that observed at different flowering dates, which indicated that, the key factor influencing these three indices was daily mean temperature during 050 days post anthesis (DPA) formed at different flowering dates. Our result was also showed that there is an interaction between nitrogen application rate and flowering date. Increasing application of N could reduce the variation of these indexes formed at flowering dates because nitrogen application rates could significantly affect the process of these three cotton fiber quality indices through influencing cotton boll subtending leaf N concentration (NA).