Updated project metadata. - Pollen tube growth is important process for successful double fertilization, which is critical for grain yield in crop plants. Despite much progress in identification of rapid alkalization factors (RALFs) which serve as ligand for signaling transduction during fertilization in Arabidopsis, there is no functional study of RALF in mono-cotyledon plant. - We functionally characterized two pollen specific RALF in rice (Oryza sativa) using multiple CRISPR/Cas9 induced loss-of-function mutants, peptide treatment, expression analyses, tag reporter lines. - OsRALF17 is specifically expressed in pollen and pollen tube as the strongest level among 41 RALF members in rice. Exogenously applied OsRALF17 inhibits pollen tube germination and elongation at high concentration, but enhances tube elongation at low concentration, indicating the regulation of growth balance. Double mutant of OsRALF17 with OsRALF19 exhibit almost male sterile, with defect on pollen germination and tube elongation. - Our study revealed that functionally-redundant OsRALF17 and 19 peptides binds to the OsMTD2, CrRLK1L family member, and transmits ROS signal for pollen tube germination and integrity maintenance in rice. We provide new insights into the role of RALF and expanding our understanding of the biological role of RALF in regulating rice fertilization.