β-Cell apoptosis occurs in diabetes mellitus (DM). Heat shock protein (HSP) 27 (human homolog of rodent HSP25) mitigates stress-induced apoptosis but has not been studied in β-cells. We tested whether HSP27 overexpression attenuates streptozotocin (SZ)-induced DM in vivo and cytokineinduced islet apoptosis in vitro. DM was ascertained by ip glucose tolerance testing, and fasting serum insulin/glucose was measured. Pancreas was stained for insulin, HSP27, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and insulin content was measured. HSP25/27 was measured by immunoblotting, isoelectric focusing, and RT-PCR. Islet HSP25/27 oligomerization and inhibitory κB protein kinase γ (nuclear factor κB essential modulator) binding were assessed by coimmunoprecipitation. HSP27 transgene (TG) in pancreas localized predominantly in β-cells. Baseline pancreatic insulin levels in wild-type (WT) and HSP27TG mice were similar, but lower in WT than HSP27TG after SZ (P < 0.01). Intraperitoneal glucose tolerance testing confirmed protection from SZ-DM in HSP27TG. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and inducible nitric oxide synthase staining were increased inWTvs. HSP27TG islets (P<0.05) after SZ. Caspase-3 activity was lower in islets from HSP27TG vs. WT mice after cytokine stress in vitro (P < 0.05). There was more HSP25 plus 27 protein from HSP27TG islets than HSP25 from WT (P < 0.01). HSP25 protein but not mRNA was increased in HSP27TG mice. Isoelectric focusing showed similar relative HSP phosphorylation in HSP27TG and WT (P > 0.05). HSP27 bound native HSP25 in TG islets; both bound to inhibitory κB protein kinase γ (nuclear factor κB essential modulator). These data show islet protection by HSP27 by mitigation of apoptosis, possibly through nuclear factor κB regulation. Copyright © 2009 by The Endocrine Society.