Diagnosing the status of crop nitrogen (N) helps to optimize crop yield, improve N use efficiency, and reduce the risk of environmental pollution. The objectives of the present study were to develop a critical N (Nc) dilution curve for winter wheat (based on spike dry matter [SDM] during the reproductive growth period), to compare this curve with the existing Nc dilution curve (based on plant dry matter [DM] of winter wheat), and to explore its ability to reliably estimate the N status of winter wheat. Four field experiments, using varied N fertilizer rates (0–375 kg ha-1) and six cultivars (Yangmai16, Ningmai13, Ningmai9, Aikang58, Yangmai12, Huaimai 17), were conducted in the Jiangsu province of eastern China. Twenty plants from each plot were sampled to determine the SDM and spike N concentration (SNC) during the reproductive growth period. The spike Nc curve was described by Nc = 2.85×SDM-0.17, with SDM ranging from 0.752 to 7.233 t ha-1. The newly developed curve was lower than the Nc curve based on plant DM. The N nutrition index (NNI) for spike dry matter ranged from 0.62 to 1.1 during the reproductive growth period across the seasons. Relative yield (RY) increased with increasing NNI; however, when NNI was greater than 0.96, RY plateaued and remained stable. The spike Nc dilution curve can be used to correctly identify the N nutrition status of winter wheat to support N management during the reproductive growth period for winter wheat in eastern China.