Trimethylamine N-oxide (TMAO) impairs β-cell function and glucose tolerance in both mice and humans. TMAO levels are elevated in diabetic subjects and mice, and they inhibit glucose-stimulated insulin secretion (GSIS) in MIN6 cells and primary islets. In male C57BL/6J mice, elevated TMAO levels impair GSIS, β-cell proportion, and glucose tolerance. TMAO inhibits GSIS by reducing calcium transients through NLRP3 inflammasome-related cytokines and inducing Serca2 loss. Long-term TMAO exposure promotes β-cell ER stress, dedifferentiation, and apoptosis, and inhibits β-cell transcriptional identity. Inhibition of TMAO production improves GSIS, β-cell proportion, and glucose tolerance in *db/db* and choline diet-fed mice. These findings suggest that TMAO plays a crucial role in β-cell dysfunction and maintenance, and inhibiting TMAO could be a potential therapeutic approach for type 2 diabetes (T2D).Trimethylamine N-oxide (TMAO) impairs β-cell function and glucose tolerance in both mice and humans. TMAO levels are elevated in diabetic subjects and mice, and they inhibit glucose-stimulated insulin secretion (GSIS) in MIN6 cells and primary islets. In male C57BL/6J mice, elevated TMAO levels impair GSIS, β-cell proportion, and glucose tolerance. TMAO inhibits GSIS by reducing calcium transients through NLRP3 inflammasome-related cytokines and inducing Serca2 loss. Long-term TMAO exposure promotes β-cell ER stress, dedifferentiation, and apoptosis, and inhibits β-cell transcriptional identity. Inhibition of TMAO production improves GSIS, β-cell proportion, and glucose tolerance in *db/db* and choline diet-fed mice. These findings suggest that TMAO plays a crucial role in β-cell dysfunction and maintenance, and inhibiting TMAO could be a potential therapeutic approach for type 2 diabetes (T2D).