Pollen fertility is an agronomic trait that strongly influences rice yield. Recent studies have revealed that the development of the pollen wall is required for pollen fertility and is regulated by several genes. However, the mechanisms underlying pollen and pollen wall development in rice remain largely unknown. In the present study, a point mutation in a gene on chromosome 1 was identified that resulted in the production of less and shrunken pollen (LSP) and led to defects in pollen wall formation. This gene was named LSP1 and was found to encode a member of the adenosine triphosphate-binding cassette (ABC) transporter G subfamily, OsABCG3. Two other loss-of-function mutants of LSP1/OsABCG3, generated using CRISPR/Cas9 technology, showed the same male sterile phenotype. The LSP1/OsABCG3 gene showed a spatio-temporal expression patt in the developing anthers, and is an ortholog of the Arabidopsis genes AtABCG1 and AtABCG16, which play an important role in pollen wall development. Mutation of LSP1/OsABCG3 affected the expression of several genes involved in pollen and pollen wall formation. These results suggest that LSP1/OsABCG3 is critical for normal pollen fertility and shed light on the molecular mechanisms underlying rice pollen wall development.