Deficiency of copper (Cu) causes low fertility in many plant species, but the molecular mechanisms underlying distribution of Cu to the floral organs are poorly understood. Here, we found that a member of yellow-stripe like (YSL) family, YSL16 encoding the Cu-nicotianamine (Cu-NA) transporter, was highly expressed in the rachilla, with less expression in the palea and lemma of rice (Oryza sativa). β-Glucuronidase (GUS) staining of transgenic rice carrying the OsYSL16 promoter-GUS showed that OsYSL16 was mainly expressed in vascular bundles of the rachilla as well as the palea and lemma. Knockout of OsYSL16 resulted in decreased Cu distribution to the stamens, but increased distribution to the palea and lemma. A short-term (24 h) 65Cu labeling experiment confirmed increased Cu concentration of palea and lemma in the mutant. Furthermore, we found that redistribution of Cu from the palea and lemma was impaired in the osysl16 mutant after exposure to Cu-free solution. The osysl16 mutant showed low pollen germination, but this was rescued by addition of Cu in the medium. Our results indicate that OsYSL16 expressed in the vascular bundles of the rachilla is important for preferential distribution of Cu to the stamens, while OsYSL16 in vascular bundles of the palea and lemma is involved in Cu redistribution under Cu-limited conditions in rice.