Acrolein is an identified high-priority hazardous air pollutant ubiquitous in daily life and associated with cardiometabolic risk that attracts worldwide attention. However, the etiology role of acrolein exposure in glucose dyshomeostasis and type 2 diabetes (T2D) is unclear. This repeated-measurement prospective cohort study included 3522 urban adults. Urine/blood samples were repeatedly collected for determinations of acrolein metabolites (N-acetyl-S-(3-hydroxypropyl)-l-cysteine, N-acetyl-S-(2-carboxyethyl)-l-cysteine; acrolein exposure biomarkers), glucose homeostasis, and T2D at baseline and a three-year follow-up. We found that each 3-fold increment in acrolein metabolites was cross-sectionally associated with 5.91-6.52% decrement in homeostasis model assessment-insulin sensitivity (HOMA-IS) and 0.07-0.14 mmol/L, 4.02-4.57, 5.91-6.52, 19-20, 18-19, and 23-31% increments in fasting glucose (FPG), fasting insulin (FPI), HOMA-insulin resistance (HOMA-IR), risks of prevalent IR, impaired fasting glucose (IFG), and T2D, respectively; longitudinally, participants with sustained-high acrolein metabolite levels had increased risks of incident IR, IFG, and T2D by 63-80, 87-99, and 120-154%, respectively (P < 0.05). In addition, biomarkers of heme oxygenase-1 activity (exhaled carbon monoxide), lipid peroxidation (8-iso-prostaglandin-F2α), protein carbonylation (protein carbonyls), and oxidative DNA damage (8-hydroxy-deoxyguanosine) mediated 5.00-38.96% of these associations. Our study revealed that acrolein exposure may impair glucose homeostasis and increase T2D risk via mediating mechanisms of heme oxygenase-1 activation, lipid peroxidation, protein carbonylation, and oxidative DNA damage.