The Rho GDP dissociation inhibitor gene OsRhoGDI1 was isolated as the putative partner of OsRac5 from rice young panicles by the yeast two-hybrid screening, but the characterization and functions of the gene remains unknown. In this study, on the basis of the bioinformatics analysis, expression profile chip mining and RT-qPCR were employed to analyze the expression pattern of OsRhoGDI1, and it was found that the gene was widely expressed in various tissues of rice, especially with high abundance during the development of young panicles, and its expression was regulated by abscisic acid (ABA), brassinolide (BL), salicylic acid (SA), indole-3-acetic acid (IAA), gibberellin (GA) and high salinity. Subcellular localization identification showed that OsRhoGDI1 was distributed in the cytomembrane, cytoplasm, and nucleus. Bimolecular fluorescence completion (BiFC) was further performed to identify the in vivo interaction between OsRhoGDI1 and OsRac5, the results suggested that the interaction occurred in the nucleus, cytomembrane, and cytoplasm. In addition, OsRhoGDI1 interacted with OsRac1 in the cytomembrane. Our results indicated that OsRhoGDI1 may be involved in rice panicle development, and participate in regulating various biological processes such as rice growth and development, hormone response, and abiotic stress by regulating the activities of different Rho/Rop proteins via interactions.