MIP-1α, a CC chemokine, recruits monocytes, natural killer cells, lymphocytes, and neutrophils, and plays a critical role in viral infection. Since, the lesional epidermis of herpes zoster expressed MIP-1α, we hypothesized that keratinocytes produce MIP-1α in response to virus-associated dsRNA via TLR3. To investigate this, we examined cultured human keratinocytes for MIP-1α production induced by poly(I:C), a TLR3 ligand. Poly(I:C) treatment induced MIP-1α production, interestingly, poly(I:C)-induced IFN-α and -β production preceded MIP-1α production. A neutralizing antibody for IFN-β significantly inhibited the poly(I:C)-induced MIP-1α production indicating that MIP-1α production is via IFN-β. IFN-α priming enhanced TLR3 expression and MIP-1α production in poly(I:C)-treated keratinocytes. This suggests that IFN-α enhanced the TLR3 expression and reinforced the response of keratinocytes to poly(I:C), which resulted in an increase in MIP-1α production. In conclusion, normal human keratinocytes produce MIP-1α in response to dsRNA via TLR3, and this production is regulated by IFN-α/β.