Trimethylamine N-oxide (TMAO) impairs β-cell function and glucose tolerance. TMAO, found at levels similar to those in diabetes, reduces glucose-stimulated insulin secretion (GSIS) in β-cells and primary islets from mice and humans. In male C57BL/6J mice, elevated TMAO levels impair GSIS, β-cell proportion, and glucose tolerance. TMAO inhibits calcium transients via the NLRP3 inflammasome and reduces Serca2, which is reversed by a Serca2 agonist. Long-term TMAO exposure causes β-cell ER stress, dedifferentiation, and apoptosis, and inhibits β-cell transcriptional identity. Inhibiting TMAO production improves β-cell function and glucose tolerance in db/db and choline diet-fed mice. TMAO is associated with diabetes and is produced by gut microbiota metabolizing dietary factors like choline. TMAO increases in diabetes and exacerbates insulin resistance. Knockdown of Fmo3, the TMAO-producing enzyme, prevents insulin resistance and obesity. TMAO inhibits mitochondrial respiration and cytosolic calcium transients, leading to β-cell dysfunction. TMAO reduces Serca2 expression through the NLRP3 inflammasome, impairing β-cell function. Long-term TMAO exposure promotes β-cell dedifferentiation and apoptosis. Inhibiting TMAO via Fmo3 knockdown improves β-cell function and glucose tolerance in db/db mice. These findings suggest TMAO is a key factor in β-cell dysfunction and T2D, and targeting TMAO could be a therapeutic approach for T2D.Trimethylamine N-oxide (TMAO) impairs β-cell function and glucose tolerance. TMAO, found at levels similar to those in diabetes, reduces glucose-stimulated insulin secretion (GSIS) in β-cells and primary islets from mice and humans. In male C57BL/6J mice, elevated TMAO levels impair GSIS, β-cell proportion, and glucose tolerance. TMAO inhibits calcium transients via the NLRP3 inflammasome and reduces Serca2, which is reversed by a Serca2 agonist. Long-term TMAO exposure causes β-cell ER stress, dedifferentiation, and apoptosis, and inhibits β-cell transcriptional identity. Inhibiting TMAO production improves β-cell function and glucose tolerance in db/db and choline diet-fed mice. TMAO is associated with diabetes and is produced by gut microbiota metabolizing dietary factors like choline. TMAO increases in diabetes and exacerbates insulin resistance. Knockdown of Fmo3, the TMAO-producing enzyme, prevents insulin resistance and obesity. TMAO inhibits mitochondrial respiration and cytosolic calcium transients, leading to β-cell dysfunction. TMAO reduces Serca2 expression through the NLRP3 inflammasome, impairing β-cell function. Long-term TMAO exposure promotes β-cell dedifferentiation and apoptosis. Inhibiting TMAO via Fmo3 knockdown improves β-cell function and glucose tolerance in db/db mice. These findings suggest TMAO is a key factor in β-cell dysfunction and T2D, and targeting TMAO could be a therapeutic approach for T2D.