Purpose <p>Evaluating the effect of novel plant growth regulator (mixture of 27% ethephon (2-chloroethyl-phosphonic acid) and 3% diethyl aminoethyl hexanoate (DA-6)) and nitrogen (N) application on photosynthesis, endogenous hormones content and grain filling in winter wheat is crucial for maximum wheat production. The purpose of the experiment was to improve grain filling in winter wheat.</p> Methods <p>We conducted an experiment to investigate the effect of novel plant growth regulator i.e., 0 mL ha^–1 (E₀, water spray) and 400 mL ha^–1 (E₄₀₀) and N application i.e., 0&#xa0;kg N ha^–1 (N₀), 200&#xa0;kg N ha^–1 (N₂₀₀) and 300&#xa0;kg N ha^–1 (N₃₀₀) on photosynthesis, endogenous hormones content and its relation with grain filling in winter wheat.</p> Results <p>Novel plant growth regulator and optimum (N₂₀₀) N application significantly increased abscisic acid, zeatin and zeatin riboside content in superior and in inferior grains in winter wheat. The higher abscisic acid, zeatin and zeatin riboside content significantly increased grain weight and grain filling rate in superior and in inferior grains in winter wheat. Novel plant growth regulator and optimum N application significantly increased grain yield (by 78.13% on average) in winter wheat compared with the E₀N₀. The increase in grain yield was attributed to higher grain weight and grain filling rate in superior and in inferior grains in winter wheat. In addition, treatment E₄₀₀N₂₀₀ increased leaf area (by 39.11%), shoot dry matter per plant (by 48.63%), chlorophyll content and gas exchange parameters in winter wheat compared with the E₀N₀. The plant hormone abscisic acid, zeatin and zeatin riboside contents have a significant positive correlation with grain filling characteristics.</p> Conclusions <p>In conclusion, novel plant growth regulator and optimum (N₂₀₀) N application resulted in higher grain yield in winter wheat by improving the photosynthesis and grain filling.</p>