Digital dermatitis is a painful foot disease compromising welfare in dairy cattle. The disease has a complex multibacterial aetiology, but little is known about its pathogenesis. In this study, gene expression in skin biopsies from five bovine digital dermatitis lesions and five healthy bovine feet was compared using RNA-Seq technology. Differential gene expression was determined after mapping transcripts to the Btau 4.0 genome. Pathway analysis identified gene networks involving differentially expressed transcripts. Bovine digital dermatitis lesions had increased expression of mRNA for a2-macroglobulin-like 1, a protein potentially involved in bacterial immune evasion and bacterial survival. There was increased expression of keratin 6A and interleukin 1b mRNA in bovine digital dermatitis lesions, but reduced expression of most other keratin and keratin-associated genes. There was little evidence of local immune reactions to the bacterial infection present in lesions. Ten samples were processed; five normal skin biopsies from the hind foot skin and five digital dermatitis lesions (large (>2cm) red raw in appearance).