<p>Syntaxin-4, a beta cell plasma membrane-anchored protein, attaches to beta cell granules with two spatially-located nearby proteins, facilitating the fusion of beta cell-docked granules to the plasma membrane, followed by exocytotic release of insulin and remaining granule contents. During type 1 diabetes (T1D) and its preceding phase, it is unclear whether syntaxin-4 levels decline in residual beta cells, thereby contributing to impaired insulin release. The level of syntaxin-4 in insulin-positive regions of islets was determined following immunofluorescence staining of human pancreatic sections, with co-immunofluorescence staining for insulin and for glucagon by immunoperoxidase. Random islets from non-diabetic autoantibody-negative (Group 1; <i>n</i> = 7), non-diabetic autoantibody-positive (Group 2; <i>n</i> = 6), new-onset T1D (Group 3; <i>n</i> = 4) and long-term T1D (Group 4; <i>n</i> = 10) donors were quantified for syntaxin-4 levels. In groups 1 and 2, inter-islet staining intensities for syntaxin-4, although variable across individual donors, were similar overall between the groups, with lower intensities in group 3. In group 4, 3 donors with 0.58–1.5 years of T1D showed mean intensities comparable to groups 1 and 2, whereas donors with 4–7 years of T1D showed declining intensities. In general, the average syntaxin-4 intensities in the two diabetic groups were lower than those in the non-diabetic groups. The expression of syntaxin-4 in insulin-positive islet cells showed marked differences in intensity among most diabetic donors and autoantibody-positive and -negative non-diabetic donors. In some long-term diabetic donors, syntaxin-4 was also present in insulin- and glucagon-negative cells. During protracted T1D, reduced syntaxin-4 in islets may impair insulin release.</p>