Small RNAs are a class of non-coding RNAs that typically range from 20 to 24 nucleotides in length. Among them, microRNAs (miRNAs) are particularly important regulators for plant development. The biological function of the conserved miRNAs has been studied extensively in plants, while that of the species-specific miRNAs has been studied in-depth. In this study, the regulatory role of a rice-specific OsmiRNA5488 (OsmiR5488) was characterized with the miR5488-overexpressed line () and miR5488-silenced line (). The seed-setting rate was notably reduced in lines, but not in lines. Cytological observation demonstrated the different types of abnormal mature embryo sacs, including the degeneration of embryo sacs and other variant types, in lines. The percentage of the abnormal mature embryo sacs accounted for the reduced value of the seed-setting rate. Furthermore, was identified as a target of via RNA ligase-mediated 3'-amplifification of cDNA ends, dual luciferase assays, and transient expression assays. The primary root length was decreased with the increases in auxin concentrations in lines compared to wild-type rice. Summarily, our results suggested that OsmiR5488 regulates the seed-setting rate and down-regulates the targeted gene .