Previous reports indicated that AtMYB103 has an important role in tapetum development, callose dissolution, and exine formation in A. thaliana anthers. Here, we further characterized its function in anther development by expression pattern analysis, transmission electron microscopy observation of the knockout mutant, and microarray analysis of downstream genes. A total of 818 genes differentially expressed between ms188 and the wild-type were identified by global expression profiling analysis. Functional classification showed that loss-of-function of AtMYB103 impairs cell wall modification, lipid metabolic pathways, and signal transduction throughout anther development. RNA in situ hybridization confirmed that transcription factors acting downstream of AtMYB103 (At1g06280 and At1g02040) were expressed in the tapetum and microspores at later stages, suggesting that they might have important roles in microsporogenesis. These results indicated that AtMYB103 is a crucial regulator of Arabidopsis anther development.