Islet transplantation, an effective treatment for Type I Diabetes, is compromised by transient ischemia. Ischemia reduces the viability and function of transplanted islets and may also promote signals that lower islet efficacy. In order to characterize the response of human islets to transient ischemia, high-throughput sequencing was employed to determine global gene expression. Islets isolated from healthy donors (n=4) were either pelleted in microcentrifuge tubes (ischemia) or cultured in vessels with oxygen-permeable membranes for 12 h at 37 °C, ambient O2, and 5% CO2 in a paired fashion. Isolated mRNA (n=4) was sequenced using Illumina RNAseq and analyzed for differentially expressed transcripts with edgeR. RNA transcripts were queried for enrichment and modelled to functional pathways previously defined using Ingenuity IPA and public databases. Network analysis of differentially expressed genes was performed to measure the connectivity of protein products in physical space to iterate functional subnetworks. RNAseq identified over 22 000 transcripts of which 650 were differentially expressed (FDR<0.05) in ischemic islets. Eight canonical pathways were enriched (corrected P<0.05), with the highest scoring pathways; TNF signalling, HIF-1alpha transcription factor network, and cellular senescence. Upstream analysis with IPA identified IL1B, TNF, TGBF1, and PDGF BB (activation z-score >6) as upstream regulators based on the differentially expressed genes. Network analysis revealed highly connected sub-networks involved in cell death, injury and tissue morphology, and amino acid metabolism. These findings are consistent with expectations following ischemic insult, however illustrate the dynamic response of cell clusters to hypoxia and potentially nutrient deprivation. Isolated human islets exposed to ischemia had differentially expressed cytokine profiles consistent with increased inflammation. Ischemia also activated TNF and likely IL1B, both of which are established stress response and survival signals in islets. Identification of tangible molecular targets provides the framework for intervention strategies in order to improve islet health during isolation and transplantation procedures.