Abstract: A high grain number per spike is the basis for achieving a high yield. Promoting spike and floret development, and reducing floret degeneration and abortion can effectively increase the development of fertile florets into more grains. Exploring the effect of spraying exogenous 14-hydroxybrassinosterol (14-HBR) on the development of wheat florets into grains contributes to increasing grains number per spike and improving yield. A field experiment of spraying 0.05 μmol∙L⁻¹ 14-HBR on wheat leaf surface at 20 days after jointing was conducted with multi-spike wheat cultivar ‘Yumai 49-198’ and large-spike wheat cultivar ‘Zhoumai 16’. The development of young spikes and florets, carbon and nitrogen metabolism enzymes in flag leaves, dry matter weight, soluble sugar and nitrogen contents of different organs, and yield components were measured to analyze the effect of exogenous 14-HBR on the optimization of spike and floret development, as well as the physiological mechanism of 14-HBR promoting grains number. The results showed that spraying exogenous 14-HBR increased the number of fertile florets and the setting rate of fertile florets and spikelets. The promoting effect was more obvious for the large-spike wheat cultivar ‘Zhoumai 16’. Spraying exogenous 14-HBR increased the activities of RuBisCo enzyme, sucrose phosphorylase, and sucrose synthase for carbon metabolism, and activities of glutamine synthetase and nitrate reductase for nitrogen metabolism in wheat flag leaf, which promoted the synthesis of photosynthetic products and the metabolism and accumulation of carbon and nitrogen. Overall, the promoting effect was higher for large-spike wheat cultivar ‘Zhoumai 16’ than multi-spike wheat cultivar ‘Yumai 49-198’. Spraying exogenous 14-HBR increased the contents of soluble sugar and nitrogen and dry matter accumulation of spike organ and non-spike organ, promoted the transport and distribution of soluble sugar to spike, and improved the C/N ratio of spike organs, which provided sufficient carbon nutrients for spike and floret development, and overall, the promoting effect was more effective for large-spike wheat cultivar ‘Zhoumai 16’. To analyze the effect of exogenous 14-HBR on the yield and yield components, spraying 14-HBR 20 days after elongation had little effect on spike number and thousand-grain weight, but a significant impact on grains number per spike and yield was observed. Spraying exogenous 14-HBR significantly improved the grains number per spike, which was the main way to increase the yield. Compared with CK treatment (no spraying 14-HBR but spraying water), for the spraying 14-HBR treatment of multi-spike wheat cultivar ‘Yumai 49-198’ and large-spike wheat cultivar ‘Zhoumai 16’, the increase in grains number per spike was 9.85% and 11.40%, respectively, and the increase in yield was 9.31% and 12.03%, respectively. Spraying exogenous 14-HBR mainly improved the carbon assimilation ability of wheat, enhanced nitrogen absorption and accumulation, and promoted the transport and distribution of carbon nutrients to spike organs, thereby providing a good material basis for the two-stage differentiation of florets, reducing the degradation and abortion of florets, and increasing the number and rate of floret settings.