Neo-tetraploid rice, a type of high-fertility tetraploid rice, is thought to be a useful material for utilizing its heterosis. However, the mechanism of its high fertility remains little known. Here, two tetraploid fertility-directed lines were generated and used to evaluate their variation on fertility charts. Cytological observations indicated that the low tetraploid fertility-directed lines (LFDL) exhibited a lower seed-setting ratio (12.64 %), pollen fertility (53.28 %), embryo sac fertility (85.71 %) while compared with the high tetraploid fertility-directed lines (HFDL). In addition, approximately 31.44 % and 48.13 % of chromosome lagging at Metaphase I and Metaphase II, and 33.33 % and 53.47 % of chromosome straggling at Anaphase I and Anaphase II were detected in the LFDL, respectively. Transcriptome analysis identified 911 differentially expressed genes (DEGs) in the HFDL compared with the LFDL. Among these DEGs, 202 meiosis-related or stage-specific genes exhibited significant down-regulation in HFDL compared with LFDL. Further, we selected NTFR1 as the candidate gene and verified its fertility phenotype in knock-out mutants, and detected a significant decrease in the seed-setting ratio, pollen viability, pollen fertility, and embryo sac fertility. This study provided a new fertility gene for tetraploid rice, and it may offer the fertility regulatory mechanisms in neo-tetraploid rice.