Downregulation of transcription factor AtMYB103 using transgenic technology results in early tapetal degeneration and pollen aberration in the anther development in Arabidopsis thaliana. In this paper, we performed further functional analysis of this gene based on three knockout mutants. Ms188-1 and ms188-2 were screened using an ethyl methane sulfonate (EMS) mutagenesis. Both mutants are male sterile. A map-based cloning approach was used and ms188 was mapped to a region of 95.8kb on chromosome 5. This region contains a transcription factor AtMYB103. Sequence analysis revealed that ms188-1 has a premature stop codon in the coding region of AtMYB103. A transposon-tagging line of AtMYB103 bought from RIKEN (Japan) also shows male sterile phenotype. The test of allelism indicated that MS188 and AtMYB103 belong to the same locus. Cytological observation showed the tapetum development was defected, and callose dissolution was affected in ms188 plants. Most microspores in matured anthers are degraded with surviving microspores lacking exine in ms188 mutant. AtMYB103 encodes a R2R3 MYB protein and is located predominantly in the nucleus. Realtime-PCR analysis indicated that the callase related gene A6 was downregulated, and the expression of exine formation gene MS2 can not be detected in the mutant anther. All these results suggest that AtMYB103 regulate tapetum development, callose dissolution and exine formation in Arabidopsis thaliana.