Chloroplasts, essential organelles for photosynthesis, play a crucial role in plant growth and productivity. Chloroplast development is regulated by the transcription of chloroplast genes, facilitated by plastid-encoded RNA polymerase (PEP). Because the transcriptional activity of PEP largely relies on PEP-associated proteins (PAPs), therefore, PAPs serve as pivotal regulators in the process of chloroplast development. This study focuses on the identification and characterization of rice PAP3 (OsPAP3) and its role in chloroplast biogenesis. The expression level of OsPAP3 was much higher in leaves than in roots and was positively regulated by light, similar to the PEP-dependent chloroplast gene OsRbcL. Additionally, OsPAP3 protein localization was observed in chloroplast nucleoids where PEP functions, Homozygous OsPAP3 mutant plants exhibited an albino phenotype due to impaired chloroplast biogenesis. In the knock-out mutants, the transcription levels of PEP-dependent chloroplast genes such as OsRbcL and OsPsbA were strongly reduced compared with wild-type plants. Conversely, OsPAP3-overexpressing plants displayed increased chloroplast numbers and higher PEP-dependent chloroplast gene expression than wild-type plants. These findings suggest that OsPAP3 is essential for PEP-dependent chloroplast gene expression and chloroplast biogenesis in rice. More importantly, data from 3 seasons of field cultivation revealed that the overexpression of OsPAP3 improves rice grain yield by approximately 25%, largely due to increased tiller formation. Collectively, these suggest that OsPAP3 regulates rice growth and productivity by controlling chloroplast development.