Poor filling and low weight of inferior kernels limit the further improvement of wheat yield. Two culti-vars, Shuangda 1 and Xinong 538, with different grain weights, were selected to investigate the physio-logical changes of inferior kernels by removal of superior kernels (RS) at the flowering stage. iTRAQ combined with physiological indexes was used to identify factors limiting the filling of inferior kernels. Removal of superior kernels significantly increased the mean filling rate of inferior kernels and thus increased their weight. A set of 6012 proteins in inferior wheat kernels were differentially expressed between the RS and control. These differentially expressed proteins were involved mainly in carbon metabolism and energy metabolism. The main reason for the promoting effect of RS on the filling of infe-rior kernels may be that RS downregulated proteins involved in glycolysis and pyruvate metabolism while upregulating proteins involved in carbon fixation and photosynthesis. Consequently, RS greatly increased the ATP content in inferior kernels, supplying energy for them to absorb photosynthetic assim-ilates. Removal of superior kernels increased the activities of sucrose synthase, soluble starch synthase, adenosine diphosphate glucose pyrophosphorylase, and starch branching enzyme in inferior kernels and promoted starch accumulation in them. Thus, RS promoted the filling of inferior kernels and increased their weight. The promoting effect of RS on starch synthesis in inferior kernels was associated with their endogenous IAA and ABA levels.