Present knowledge of innate immunity in infected cells relies on studies of cell lines and animal models. In this study, primary human pancreatic islets of Langerhans were used to study virus–host interactions in a model of the possible induction of type 1 diabetes by enterovirus (EV). Human islets were infected with a strain of EV isolated at onset of type 1 diabetes, or exposed to synthetic dsRNA (poly(I:C)), used commonly to mimic viral infection. Induction of innate immunity and the effect of the female sex hormone 17β-estradiol, known to have cell-protective effects, on islet chemokine secretion were investigated. 17β-Estradiol reduced EV–but not poly(I:C)-induced IP-10/CXCL10 secretion from human islets, suggesting that separate signaling pathways of the innate immune response are triggered by EV and poly(I:C), respectively. Infection with EV increased the gene-expression of toll-like receptor 3, interferon-β, and the intracellular helicase MDA5, involved in antiviral innate immunity, multi-fold over time, whereas poly(I:C) increased the expression of these genes transiently. The induced expression pattern was similar in all donors, but the expression levels varied greatly. Pre-exposure to poly(I:C) blocked viral replication in islets from 56% of the donors. These data provide insight on the innate immune responses induced by EV in human islets, and show that this can be modulated by 17β-estradiol, and suggest an important difference between virus- and poly(I:C)-induced signaling. J. Med. Virol. 83:658–664, 2011. © 2011 Wiley-Liss, Inc.