Excessive use of nitrogen fertilizer and high planting density reduce grain weight in wheat. However, the effects of high nitrogen and planting density on the filling of grain located in different positions of the wheat spikelet are unknown. A two-year field experiment was conducted to investigate this question and the underlying mechanisms with respect to hormone and carbohydrate activity. Both high nitrogen application and planting density significantly increased spike density, while reducing kernel number per spike and 1000-kernel weight. However, the effects of high nitrogen and high plant density on kernel number per spike and 1000-kernel weight were different. The inhibitory effect of high nitrogen application and high planting density on kernel number per spike was achieved mainly by a reduction in kernel number per spikelet in the top and bottom spikelets. However, the decrease in 1000-kernel weight was contributed mainly by the reduced weight of grain in the middle spikelets. The grain-filling rate of inferior grain in the middle spikelets was significantly decreased under high nitrogen input and high planting density conditions, particularly during the early and middle grain-filling periods, leading to the suppression of grain filling and consequent decrease in grain weight. This effect resulted mainly from inhibited sucrose transport to and starch accumulation in inferior grain in the middle spikelets via reduction of the abscisic acid/ethylene ratio. This mechanism may explain how high nitrogen application and high planting density inhibit the grain filling of inferior wheat grain.