Panicle apical abortion occurs frequently in rice production, which can affect grain number per panicle and when serious may lead to the reduction of grain yield. Although some rice panicle apical abortion genes such as PAA1, PAA2, and TUT1 have been identified, their underling molecular mechanism are still poorly understood. In this study, we report a gene mapping and cloning of a recessive PAA tutou3 mutant in rice. The mutant had shorter plant height during vegetative development stage and serious panicle apical abortion at the later stage of panicle development, which resulted in significantly reduced grain number per panicle, panicle length and 1000-grain weight. Genetic analysis demonstrated that the phenotype of tutou3 was determined by a single recessive gene. Using 856 individuals with serious panicle apical abortion in the F2 segregating population derived from tutou3 × IRAT129, TUTOU3 locus was mapped in a 29 kb interval flanked by markers A-2 and A-17 on chromosome 4 containing 3 putative open reading frames (ORFs). Sequence analysis revealed a single nucleotide substitution of C to A at the eighth exon of LOC_Os04g56160, a plasma membrane H+-ATPase encoding gene, in tutou3, resulting in an amino acid change from alanine to aspartic acid. qRT-PCR analysis showed that TUTOU3 gene was constitutively expressed in rice, and its expression level decreased in the mutants. The mRNA expression levels of OsVPE2 and OsVPE3, which induce programmed cell death of apical panicle, increased significantly in the degraded spikelets in tutou3. Further study of TUTOU3 gene will reveal the role of H+-ATPase in the growth and development of panicle apical cells and help to elucidate the mechanism of panicle apical abortion at the cellular level.