Plants produce specialized metabolites to adapt to the ever-changing environments. Flavonoids are antioxidants essential for growth, development, and breeding with increased stress resistance in crops. However, the mechanism of the involvement of flavonoids in ultraviolet-B (UV-B) stress in rice (Oryza sativa) is largely unknown. In this study, we cloned and functionally identified a receptor-like kinase (OsRLCK160) and a bZIP transcription factor (OsbZIP48) positively regulating flavonoid accumulation through metabolite-based genome-wide association study of the flavonoid content in rice. Meanwhile, OsRLCK160 interacted with and phosphorylated OsbZIP48 to regulate the flavonoid accumulation and participate in UV-B tolerance in rice. Our study indicates the importance of applying OsRLCK160and OsbZIP48to advance the fundamental understanding of stable rice production and breed UV-B-tolerant rice varieties, which may contribute to breeding high-yield rice varieties.