The development of rice flowers and panicles critically affects grain yield and quality. LEAFY HULL STERILE1/OsMADS1, a grass-specific SEPALLATA-like MADS-box transcription factor, is essential for rice floral development and floral meristem activity maintenance. However, the mechanism through which OsMADS1 interacts with other genes to regulate floral organ identity and meristem determinacy remains unclear. In this study, we first generated OsMADS1 knockout mutants using CRISPR/Cas9. The mutant florets exhibited obvious morphological defects, which were categorized into five phenotypic classes. Yeast two-hybrid screening identified two OsMADS1-interacting proteins: OsMADS22, an STMADS11-like protein, and OsYABBY5, a YABBY transcription factor. Their physical interactions were validated both in vitro and in vivo, and were important for floral organ specification and meristem maintenance. Transcriptomic analysis revealed that OsMADS1 regulates numerous genes involved in hormone signaling and panicle/flower development. Furthermore, OsMADS1 acts together with OsMADS22 and OsYABBY5 to modulate the expression of the downstream target OsMADS55, thereby controlling rice spikelet development. Together, our results reveal that OsMADS1 executes diverse regulatory functions in floral organ specification and meristem identity by interacting with multiple developmental regulators, providing new insights into the molecular mechanisms of plant flower development.