Wheat inflorescence or spike is the reproductive organ and produce grains for living of human beings. However, mechanism underlying the spike development is still not clear. Using a positional cloning approach, we isolated the major quantitative trait locus HL1 on chromosome 2DS conditioning spike length, which encodes a nuclear-localized ribonuclease H-like domain-containing protein (RHLP). Wheat transformed with the RHLP sequence increased spike length, and the plants with successful RHLP silencing produced significantly shorter spikes. In both functional complementation and RNA interference experiments, we did not note significant difference in other traits caused by RHLP.