Background <p>Type 2 diabetes mellitus (T2DM) is one of the major diseases threatening human health. This study aims to investigate the role of the NBR2/miR-646 axis in T2DM and pancreatic β-cell function.</p> Methods <p>95 T2DM patients and 83 healthy individuals were enrolled in the study. Serum NBR2 and miR-646 expression were detected by quantitative reverse transcription polymerase chain reaction. The diagnostic value of NBR2 in T2DM was evaluated using receiver operating characteristic curve. To assess cell viability and apoptosis, the cell counting kit-8 assay and flow cytometry were utilized. Enzyme-linked immunosorbent assay was employed to determine insulin secretion, levels of inflammatory factors, and oxidative stress. The interaction between NBR2 and its target was confirmed using luciferase reporter assay, and their association was evaluated through Pearson correlation analysis.</p> Results <p>Serum NBR2 expression was upregulated in T2DM patients, while miR-646 expression was decreased. NBR2 could serve as a biomarker for T2DM diagnosis, with an area under the curve of 0.908 (95% confidence interval : 0.866–0.950), sensitivity of 89.5%, and specificity of 79.5%, and was negatively correlated with miR-646. Inhibiting NBR2 could increase the viability of INS-1 cells, insulin secretion, and superoxide dismutase activity, while reducing apoptosis, as well as the levels of interleukin-6, tumor necrosis factor-α, and malondialdehyde. miR-646 was identified as a target of NBR2. Inhibiting miR-646 could partially reverse the protective effect of NBR2 inhibition on cell function damage.</p> Conclusion <p>NBR2 is highly expressed in T2DM patients and has potential value in T2DM diagnosis. NBR2 can participate in the regulation of pancreatic β-cell function by modulating miR-646.</p>