Over exploitation of mineral resources has increasingly caused serious heavy metal contamination such as chromium (Cr). Cr(VI), the pathogenicity factor, is one of common environmental contaminants and widely known health hazards to living organisms. Therefore, it is urgent to control the polluted soil. Up to now, little is known about the regulatory mechanisms of Cr response in Pseudomonas sp. Cr13. In this study, transcriptome and differentially expressed genes in Pseudomonas sp. Cr13 strain was characterized by a comparison between Cr(VI)-treated sample and control sample using transcriptome sequencing approach. In total, 2974 genes were annotated, including 1245 (1154 down-regulated genes and 91 up-regulated genes) differentially expressed genes (DEGs). All DEGs could be assigned to 29 pathways, of which pathways related to amino acid metabolism, carbohydrate metabolism, energy metabolism and signal transduction mechanism were significantly enriched in Pseudomonas sp. Cr13. A possible mechanism for Cr toxicity response might be an active efflux which utilized a heavy metal translocating P-type ATPase to lower the intracellular Cr concentration. The down-regulated genes related to the antioxidant defense system had a key role in Cr reduction, such as SodA, Gst, osmC, BtuE, KatE, csdA and AhpC. The proteins that were visibly up-regulated, were likely to involve in alleviating Cr(VI) stress, and the significantly down-regulated genes such as MarR, Lrp, FhlA, GntR, HrcA, LysR family genes, were likely to reduce Cr(VI) induced oxidative stress. In addition, real-time quantitative PCR was used to analyze the expression patterns of some Cr responsive genes. This study reported the first identification of Cr responsive genes, and inferred the underlying regulatory mechanisms of response to Cr(VI) stress in Pseudomonas sp. Cr13.