Large-panicle rice cultivars often fail to reach their yield potential due to the poor grain filling of inferior spikelets (IS). Thus, it is important to determine the causes of poor IS grain filling. In this study, we attempted to identify whether inferior grain filling of large panicles is restricted by superior spikelets (SS) and their physiological mechanism. SS were removed from two homozygous japonica rice strains (W1844 and WJ165) during flowering in an attempt to force photosynthate transport to the IS. We measured the effects of SS removal on seed setting rate, grain weight, grain filling rate, sucrose content, as well as hormone levels, activities of key enzymes, and expression of genes involved in sucrose to starch metabolism in rice IS during grain filling. The results showed that SS removal improved IS grain filling by increasing the seed setting rate, grain weight, sucrose content, and hormone levels. SS removal also enhanced the activities of key enzymes and the expression levels of genes involved in sucrose to starch metabolism. These results suggest that sucrose and several hormones act as signal substances and play a vital role in grain filling by regulating enzyme activities and gene expression. Therefore, IS grain filling is restricted by SS, which limit assimilate supply and plant hormones, leading to poor grain filling of IS.