Partial rootzone irrigation (PRI) means only part of the root system is exposed to watering at one round of irrigation while the rest part is left in drying soil. The method has been proved a water-saving irrigation without much reduction in yield. This study investigated how the biomass distribution and reproductive development of cotton are affected under PRI. A three-year field irrigation experiment was conducted with a 30% reduction in irrigation amount on cotton in an arid area of Xinjiang in northwest China. Three treatments included conventional furrow irrigation (CFI) as control, alternative furrow irrigation (AFI) and fix furrow irrigation (FFI). PRI decreased stomatal conductance on the days just after irrigation when cotton plants were not under water stress, but there was no difference in stomatal conductance among irrigation treatments when plants were under water stress on the days just before next irrigation. Non-hydraulic signals from the dried rootzone inhibited the stomatal opening under well watered condition, but the moderate water deficit developed in the shoots under PRI may have played a more important role in biomass allocation and yield formation. This moderate water stress reduced shoot biomass accumulation and increased root biomass. While the vegetative and reproductive parts of the shoot were reduced in the same proportion under the PRI, the final yield was much less reduced in PRI, indicating an increased reproductive efficiency of cotton. Furthermore, PRI advanced the development of the reproductive organs and led to earlier flowering. The early matured bolls produced seed-cotton yield with a higher market value. AFI plants consistently performed better than FFI in the 3 years. We conclude that AFI can be used as a better deficit irrigation method with positive regulative effects on stomatal opening and yield forming process.