Cytokines are upregulated in prediabetes, but their relationship with Enterovirus (EV) infection and development of islet autoimmunity is unknown. Cytokines (n = 65) were measured using Luminex xMAP technology in a nested case-control study of 67 children with a first-degree relative with type 1 diabetes: 27 with islet autoantibodies (Ab⁺) and 40 age-matched persistently autoantibody negative (Ab⁻) control subjects. Of 74 samples, 37 (50%) were EV-PCR⁺ in plasma and/or stool (EV⁺) and the remainder were negative for EV and other viruses (EV⁻). Fifteen cytokines, chemokines, and growth factors were elevated (P ≤ 0.01) in Ab⁺ versus Ab⁻ children (interleukin [IL]-1β, IL-5, IL-7, IL-12(p70), IL-16, IL-17, IL-20, IL-21, IL-28A, tumor necrosis factor-α, chemokine C-C motif ligand [CCL]13, CCL26, chemokine C-X-C motif ligand 5, granulocyte-macrophage colony-stimulating factor, and thrombopoietin); most have proinflammatory effects. In EV⁺ versus EV⁻ children, IL-10 was higher (P = 0.005), while IL-21 was lower (P = 0.008). Cytokine levels did not differ between Ab⁺EV⁺ and Ab⁺EV⁻ children. Heat maps demonstrated clustering of some proinflammatory cytokines in Ab⁺ children, suggesting they are coordinately regulated. In conclusion, children with islet autoimmunity demonstrate higher levels of multiple cytokines, consistent with an active inflammatory process in the prediabetic state, which is unrelated to coincident EV infection. Apart from differences in IL-10 and IL-21, EV infection was not associated with a specific cytokine profile.