Abstract: To elucidate the regulatory roles of JrAG and JrFT genes during floral bud differentiation in walnut (Juglans regia), based on a homologous cloning strategy, the Arabidopsis AG and FT homologous genes JrAG and JrFT were isolated from female and male walnut floral buds. Overexpression vectors were constructed and transformed into Arabidopsis via Agrobacterium-mediated floral dip transformation. Transgenic lines were screened using kanamycin resistance and validated by PCR, RT-PCR, and GUS histochemical staining. Phenotypic traits—including bolting time, rosette leaf number, stamen morphology, and silique length—were systematically documented and compared between transgenic lines and wild-type (WT) plants to functionally characterize the genes. The findings reveals that the homologous genes JrAG and JrFT were successfully cloned from walnut floral buds. JrAG-overexpressing plants exhibited abnormal stamen development (filaments shortened by 42.5%, partial petaloid stamens), resulting in sterility. JrFT-overexpressing plants showed significantly earlier bolting by 6 days, accompanied by reduced rosette leaf number (8.5 → 4.8) and shorter siliques (12.5 → 8.3 mm). Molecular validation via PCR, RT-PCR, and GUS staining confirmed stable integration and tissue-specific expression of both genes in transgenic lines. Protein function was further corroborated by their expression in rosette leaves and inflorescences. These results collectively highlight the distinct yet complementary roles of JrAG and JrFT in regulating reproductive development and growth dynamics. This study identifies JrAG and JrFT as key regulators of floral development in walnut. JrAG influences reproductive development by modulating stamen morphogenesis, while JrFT alters growth rhythm by accelerating flowering time. Their synergistic action likely underpins walnut sex differentiation.