Polyploidization is one of the most effective ways to improve plant height and yield in rice (Oryza sativa L.). However, the molecular mechanism of its regulation is not yet fully understood. Here, we investigated the agronomic traits of diploid (‘Balilla-2x’) and tetraploid (‘Balilla-4x’) lines of the japonica rice variety ‘Balilla’. Compared with ‘Balilla-2x’, ‘Balilla-4x’ exhibited significantly increased plant height, spike length and yield per plant. RNA-seq analysis showed that the expression levels of yield-related genes controlling plant height and panicle development (e.g. STH1, OsYUC9 and OsDEP1) were significantly upregulated in ‘Balilla-4x’ rice plants. These results indicated that polyploidization changed the expression of genes related to agronomic traits such as plant height and spike length, thereby increasing rice yield. This study provides a further basis for understanding the yield of rice after polyploidization and can serve as a new theoretical reference for breeding high-yielding rice varieties.