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High temperature during grain filling considerably reduces yield and quality in rice, but its molecular mechanisms are not fully understood. We investigated the functions of a seed preferentially expressed Aux/IAA gene, OsIAA29, under high temperature-stress in grain filling using CRISPR/Cas9, RNAi, and overexpression. We observed that the osiaa29 had a higher percentage of shrunken and chalkiness seed, as well as lower 1000-grain weight than ZH11 under high temperature. Meanwhile, the expression of OsIAA29 was induced and the IAA content was remarkably reduced in the ZH11 seeds under high temperature. In addition, OsIAA29 may enhance the transcriptional activation activity of OsARF17 through competition with OsIAA21 binding to OsARF17. Finally, chromatin immunoprecipitation quantitative real-time PCR (ChIP-qPCR) results proved that OsARF17 regulated expression of several starch and protein synthesis related genes (like OsPDIL1-1, OsSS1, OsNAC20, OsSBE1, and OsC2H2). Therefore, OsIAA29 regulates seed development in high temperature through competition with OsIAA21 in the binding to OsARF17, mediating auxin signaling pathway in rice. This study provides a theoretical basis and gene resources for auxin signaling and effective molecular design breeding. [ABSTRACT FROM AUTHOR]