Recombinant canine interferon-γ (rCaIFN-γ) produced by a baculovirus expression system has therapeutic efficacy against atopic dermatitis in dogs. Although the mechanism of action of rCaIFN-γ is not completely understood, rCaIFN-γ is thought to downregulate the activity of interleukin-4- and interleukin-5-producing T helper 2 cells. However, rCaIFN-γ may also act directly on canine keratinocytes by inhibiting the release of inflammatory mediators. In this study, we investigated the effects of rCaIFN-γ on cytokine and chemokine mRNA transcription in a canine keratinocyte cell line, CPEK. It was found that granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA transcription was significantly inhibited after treatment with rCaIFN-γ (P < 0.001), whereas transforming growth factor-β and CC chemokine ligand 17 mRNA levels were unchanged. This study suggests that rCaIFN-γ may suppress GM-CSF production from canine keratinocytes, although further studies are required to confirm this.