Acid-Aluminum (Al) is toxic to plants and greatly affects crop production worldwide. To understand the responses of plants to acid soils and Aluminum toxicity, we examined global gene expression using microarray data in alfalfa seedlings with the treatment of acid-Aluminum. 3,926 genes that were identified significantly up- or downregulated in response to Al3+ ions with pH 4.5 treatment, 66.33% of which were found in roots. Their functional categories were mainly involved with phytohormone regulation, reactive oxygen species, and transporters. Both gene ontology (GO) enrichment and KEGG analysis indicated that phenylpropanoid biosynthesis, phenylalanine metabolism, and flavonoid biosynthesis played a critical role on defense to Aluminum stress in alfalfa. In addition, we found that transcription factors such as the MYB and WRKY family proteins may be also involved in the regulation of reactive oxygen species reactions and flavonoid biosynthesis. Thus, the finding of global gene expression profile provided insights into the mechanisms of plant defense to acid-Al stress in alfalfa. Understanding the key regulatory genes and pathways would be advantageous for improving crop production not only in alfalfa but also in other crops under acid-Aluminum stress.