Extensive research has identified enterovirus (EV) infections as key triggers of type 1 diabetes. However, the underlying molecular mechanisms via which EVs contribute to the pathogenesis of type 1 diabetes remain unclear. Given that EVs dysregulate host microRNAs (miRNAs), which function as key regulators of beta-cell biology, we investigated the impact of coxsackievrius B5 (CVB5) infection on the cellular expression of miRNAs within human islets. Using high-throughput qPCR nanofluidics arrays, the expression of 754 miRNAs were examined in CVB5-infected human pancreatic islets. In total, 33 miRNAs were significantly dysregulated (at least >3 fold difference) in the infected compared to control islets (P<0.05). Subsequently, these differentially expressed miRNAs were predicted to target mRNAs of 57 known type 1 diabetes risk genes that collectively mediate various biological processes related to diabetes, including the regulation of cell proliferation, cytokine production, and innate immune response. In conclusion, we report the first global miRNA expression profiling of CVB5-infected human pancreatic islets. We propose that EVs accelerate islet autoimmunity by disrupting the miRNA-directed suppression of pro-inflammatory factors within beta-cells, thereby resulting in an exacerbated anti-viral immune response that promotes auto-antibody production and apoptosis.