Soybean is one of the most aluminum (Al) sensitive plants. The complex inheritance of Al tolerance trait has so far undermined breeding efforts to develop Al-tolerant soybeans. Discovering the genetic factors underlying the Al tolerance mechanisms would undoubtedly accelerate the pace of such endeavor. As a first step toward this goal, we analyzed the transcriptome profile in roots of Al-tolerant soybean line PI 416937 comparing Al-treated and untreated control plants using DNA microarrays. Many genes involved in transcription activation, stress response, cell metabolism and signaling were differentially expressed. Patterns of gene expression and mechanisms of Al toxicity and tolerance suggest that Cys2His2 and ADR6 transcription activators, cell wall modifying enzymes, and phytosulfokines growth factor play role in soybean Al tolerance. Our data provide insights into the molecular mechanisms of soybean Al tolerance and will have practical value in genetic improvement of Al tolerance trait.