Dogs and humans with atopic dermatitis (AD) have a high prevalence of recurrent staphylococcal pyoderma. β-Defensins (BDs) and toll-like receptors (TLRs) are important in innate immunity against bacterial skin infections, and decreased BD and TLR2 expression has been associated with human AD. However, findings from recent studies of BD expression in human and canine AD have been variable and contradictory. The aim of this study was to further our understanding of the role of antimicrobial proteins in canine AD by quantifying mRNA for canine (c) BD1, cBD103 and TLR2 in healthy skin (n=17 dogs), matched samples of atopic skin with and without active infection by Staphylococcus pseudintermedius (n=13 dogs), and the canine keratinocyte cell line CPEK cultured with 5 ng/ml tumour necrosis factor alpha (TNFα) and 10 μg/ml lipopolysaccharide (LPS). mRNA for cBD1, CB103 and TLR2 were detected in all samples. TNFα significantly increased transcription of cBD1, cBD103 and TLR2 in the CPEK cells. mRNA for cBD103 was also significantly increased after stimulation with LPS. There were no significant differences in mRNA levels for cBD1, cBD103 or TLR2 in healthy, non-infected atopic or infected atopic skin. Canine AD did not appear to be associated with altered expression of cBD1, cBD103 and TLR2 in these dogs. Other studies have reported both increased and decreased expression of these antimicrobial peptides in canine AD and pyoderma, and therefore further investigation of the clinical significance of these mediators is required.