Introduction: IsomiRs are post transcriptional sequence variants of canonical miRNAs sequence, possibly targeting different genes. Since their biogenesis could be affected by inflammatory stress, isomiRs profiling has the potential to gain further insight into the underlying inflammatory processes within β-cells in type 1 diabetes (T1D). Methods: IsomiRs profiling was performed in Laser Capture Microdissected human pancreatic islet (LCM-HI) from n=19 non-diabetic (ND) donors in vivo metabolically characterized, EndoC-βH1 β-cell line, sorted β cells from n=3 ND donors and in collagenase-isolated human pancreatic islets (HI-Ctr) from n=3 ND donors. The impact of inflammation on isomiRs was evaluated in collagenase-isolated pancreatic islets from n=3 pre-insulitic (4 weeks) and from n=4 insulitic (8 weeks) NOD mice, and in HI treated 48h with IL-1β, TNF-α and IFN-γ (HI-Cyt). Small RNA-seq was performed. Results: IsomiRs account for a consistent percentage of miRNAs expression (59% LCM-HI, 60% HI-Ctr, 42% Sorted β-cells, 44% EndoC-βH1), with 3’end trimming representing the most abundant class (42% HI-LCM, 40% HI-Cyt, 25% Sorted β-cells, 24% EndoC-βH1). Moreover, isomiR-411 & -409, with an alternative seed sequence, are significantly associated (p<0.05), respectively, with insulin secretion rate and basal glucose, implying a functional role in human islets. A significantly increased proportion of 3’end trimmed isomiRs was observed both in insulitic NOD mice vs pre-insulitic (8w 47% vs 4w 38%; p<0.01) and in HI-Cyt vs HI-Ctr (Ctr 41%; Cyt 53%; p<0.01), revealing a link between islets inflammation and 3’end trimming. Differential proportion analysis identified n=33 miRNAs with significant increase in 3’end trimming in insulitic NOD mice and n=61 in HI-Cyt, with n=15 consistently detected in both. Conclusions: In this study (i) we profiled isomiRs in β-cells, and (ii) provide an association between increased proportion of 3’end trimmed isomiRs and inflammatory stress in beta-cell in T1D.