SummaryDownregulation of the transcription factor AtMYB103 using transgenic technology results in early tapetal degeneration and pollen aberration during anther development in Arabidopsis thaliana. This paper describes the functional analysis of the AtMYB103 gene in three knock‐out mutants. Two male sterile mutants, ms188‐1 and ms188‐2, were generated by ethyl‐methane sulfonate (EMS) mutagenesis. A map‐based cloning approach was used, and ms188 was mapped to a 95.8‐kb region on chromosome 5 containing an AtMYB103 transcription factor. Sequence analysis revealed that ms188‐1 had a pre‐mature stop codon in the AtMYB103 coding region, whereas ms188‐2 had a CCT→CTT base‐pair change in the first exon of AtMYB103, which resulted in the replacement of a proline by a leucine residue in the R2R3 domain. The third mutant, an AtMYB103 transposon‐tagging line, also showed a male sterile phenotype. Allelism tests indicated that MS188 and AtMYB103 belong to the same locus. Cytological observation revealed defective tapetum development and altered callose dissolution in ms188 plants. Additionally, most of the microspores in mature anthers were degraded and surviving microspores lacked exine. AtMYB103 encoded an R2R3 MYB protein that is predominantly located in the nucleus. Real‐time RT‐PCR analysis indicated that the callase‐related gene A6 was regulated by AtMYB103. Expression of the exine formation gene MS2 was not detected in mutant anthers. These results implicate that AtMYB103 plays an important role in tapetum development, callose dissolution and exine formation in A. thaliana anthers.