The rice (Oryza sativa L.) basic leucine Zipper factor RISBZ1 and rice prolamin box binding factor (RPBF) are transcriptional activators of rice seed storage protein (SSP) genes in vivo. To ascertain the functions of these trans-activators in seed development, knock-down (KD) transgenic rice plants were generated in which the accumulation of RISBZ1 and RPBF was reduced in an endosperm-specific manner by co-suppression (KD-RISBZ1 and KD-RPBF). The accumulation of most SSPs changed little between individual KD mutants and wild-type plants, whereas a double KD mutant (KD-RISBZ1/KD-RPBF) resulted in a significant reduction of most SSP gene expression and accumulation. The reduction of both trans-activators also caused a greater reduction in seed starch accumulation than individual KD mutants. Storage lipids were accumulated at reduced levels in KD-RISBZ1 and KD-RISBZ1/KD-RPBF seeds. KD-RPBF and KD-RISBZ1/KD-RPBF seeds exhibited multi-layered aleurone cells. Gene expression of DEFECTIVE KERNEL1 (OsDEK1), CRINKLY4 (OsCR4) and SUPERNUMERARY ALEURONE LAYER 1 (OsSAL1) rice homologues was decreased in the KD mutants, suggesting that these genes are regulated by RISBZ1 and RPBF. These phenotypes suggest that combinatorial interactions between RISBZ1 and RPBF play an essential role during grain filling. The functional redundancy and compensation between RISBZ1 and RPBF possibly account for weak effects on the SSP levels in single KD mutants, and help maintain various processes during seed development in rice. Physical interaction between RISBZ1 and RPBF may ensure that these processes are carried out properly.