Carbohydrate starvation is an efficient stress treatment for induction of microspore embryogenesis. Transcriptome analysis of the response of anthers to mannitol treatment using the 22k Barley1 GeneChip revealed large changes in gene expression. Statistical analysis and filtering for four-fold or greater changes resulted in 2673 genes, of which 887 were upregulated and 1786 downregulated. Great differences in some metabolic pathways, accompanied by a multidimensional stress response, were found. Analysis of transcription factors showed that most of the downregulated transcription factors were related to growth and development, and the upregulated factors with abiotic and biotic stress responses and changes in developmental programs. Interestingly, the expression of most cell cycle-related genes did not change significantly. Transcriptome analysis provided a successful approach for the identification of genes involved in mannitol treatment, essential for triggering microspore embryogenesis.