The objective of this study was to characterize a large portion of the bovine neutrophil transcriptome following treatment with the anti-inflammatory glucocorticoid, dexamethasone (Dex). Total RNA was isolated from blood neutrophils of healthy cattle (5 castrated male Holsteins) immediately following cell purification (0 h) or after ex vivo ageing for 4 h with or without added Dex. Additional neutrophils were co-treated with a glucocorticoid receptor (GR) antagonist (RU486) and Dex for 4 h. RNA was amplified, dye labeled (Cy3 or Cy5), and hybridized to a series of NBFGC microarrays. LOWESS data normalization followed by mixture model analyses showed that 11.15% of the spotted NBFGC cDNAs (2,036/18,263) were expressed in 4 h (untreated) neutrophils. Subsequent two-step mixed model analysis detected (P 0.05) 1109 differentially expressed genes, of which contrast analysis indicated 1064 that were independently responsive to ageing (64), Dex (502), RU486+Dex (141), or RU486 (357). In silico analysis revealed that 416 of the differentially expressed genes are unknown, 59 that did not cluster well based on known function, and 634 that clustered into 20 ontological categories. Independent validation of differential expression was done for 14 of the putatively Dex responsive genes across these categories. Results showed that Dex induced rapid translocation of GR into the neutrophil nucleus and signaled dramatic alterations in expression of genes that delay apoptosis, enhance bactericidal activity, and promote tissue remodeling without inflammation or fibrosis. Thus, these findings revealed hitherto unappreciated plasticity of blood neutrophils and potentially novel anti-inflammatory/wound healing actions of glucocorticoids.