Insulin secretion (IS) from pancreatic islet β-cells (BC) can be hampered by a stressful T1D-like microenvironment (e.g. cytokines – Cyt, increased glucose – G). We evaluated the deleterious effects of Cyt and Cyt+G on isolated human islet (HI) IS, assessed if BC dysfunction is reversible, and explored the molecular mechanisms involved. HIs from five non-diabetic organ donors (age, 78±15 yrs; BMI, 24.4±3.8 kg/m2) were exposed to Cyt (50 U/ml IL-1β + 1000 U/ml IFN-γ) at 5.5 (as in control, Ctl) or 11.1 mM G (Cyt+G) for 24h, followed by 72h culture in normal medium (wash-out, WO). IS at 3.3 and 16.7 mM G was assessed, and HI transcriptome evaluated by RNA-seq. Cyt increased IS by 2-fold at 3.3 and 16.7 mM G, with an insulin stimulation index (ISI) of 5.3±2.6 (similar to that of Ctl: 5.0±3.0). After WO, no change in IS was observed. Cyt+G decreased ISI by >50% (2.1±0.7), which improved after WO (4.0±1.3, both p<0.05), due to better IS at 16.7 mM G. Transcriptomes of Cyt vs Ctl and Cyt+G vs Ctl showed, respectively, 3,924 (padj≤0.01, 1,829 up- and 2,095 down-regulated) and 4,203 (1,998 up- and 2,205 down-regulated) differentially expressed genes (DEG). Of these, 3,281 were in common between the two comparisons, all regulated in the same direction. When the transcriptomes of Cyt+G vs Cyt treated islets were compared, 112 DEG were identified (68 up- and 44 down-regulated); GSEA retrieved 5 positively enriched pathways (including mTORC1 signaling, Unfolded protein response, Oxidative phosphorylation). After WO, Cyt+G exposed islets showed 3,137 DEG (1,443 up- and 1,694 down-regulated) and GSEA retrieved 10 positively (including Glycolysis/Gluconeogenesis, AMPK signaling, Metabolic pathways) and 65 negatively enriched pathways (including Cytokine-cytokine receptor interaction, signaling by IL-17, TNF, JAK-STAT and NF-kB, Spliceosome, Protein processing in ER). In conclusion, short-term exposure to Cyt modified HI IS, which persisted after WO. Combined Cyt+G impaired BC function, which recovered after WO. Both BC functional damage and recovery were associated with specific transcriptomic changes in metabolic processes, ER stress, inflammatory pathways, and the innate immune system. These data suggest that human BC dysfunction induced by a T1D-like environment can be reversed by alleviating the insult and/or, potentially, by targeting the underlying molecular mechanisms. Supported by IMI 2 (N. 945268; INNODIA HARVEST) and ERC (N. 866127; CAPTUR3D).