Cr(VI) is the most toxic valency form of Cr, but its toxicity targets and the cellular systems contributing to acquisition of tolerance remain to be resolved at the molecular level in plants. We used microarray assay to analyze the transcriptomic profiles of rice roots in response to Cr(VI) stress. Gene ontology analysis revealed that the 2,688 Cr-responsive genes were involved in binding activity, metabolic process, biological regulation, cellular process and catalytic activity. More transcripts were responsive to Cr(VI) during long-term exposure (24 h, 2,097 genes), than short-term exposure (1- and 3-h results pooled, 1,181 genes). Long-term Cr(VI)-regulated genes are involved in cytokinin signaling, the ubiquitin-proteasome system pathway, DNA repair and Cu transportation. The expression of AS2 transcription factors was specifically modulated by long-term Cr(VI) stress. The protein kinases receptor-like cytoplasmic kinase and receptor-like kinase in flowers 3 were significantly upregulated with only short-term Cr(VI) exposure. In addition, 4 mitogen-activated protein kinase kinase kinases, 1 mitogen-activated protein kinase (MAPK) and 1 calcium-dependent protein kinase (CDPK) were upregulated with short-term Cr(VI) treatment. Expression of reactive oxygen species and calcium and activity of MAPKs and CDPK-like kinases were induced with increasing Cr(VI) concentration. These results may provide new insights into understanding the mechanisms of Cr toxicity and tolerance during different stages in rice roots.