Animal studies suggested that exposure to ethylene oxide (EO) might induce hepatic lipid peroxidation and inflammatory lesions in various organs. However, the association between EO exposure and diabetes risk in humans is unknown. This study aimed to examine the association of EO exposure with the prevalence of diabetes mellitus in a general population of US adults. This study consisted of 3448 participants aged 20 years and older from the National Health and Nutrition Examination Survey (NHANES) 2013-2014 cycle and 2015-2016 cycle. Circulating levels of EO biomarker (hemoglobin adducts of EO (HbEO)) was measured by using high-performance liquid chromatography coupled with tandem mass spectrometry. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multivariate logistic regression. The weighted median level of HbEO was 29.9 (95% CI: 21.8, 56.0) pmol/g Hb. Elevated levels of HbEO were associated with higher HbA1c and lower high-density lipoprotein cholesterol (both P-trend <0.01). After multivariate adjustment including demographics, lifestyle factors, and body mass index (BMI), higher HbEO levels were significantly associated with an increased prevalence of diabetes mellitus. The OR (95% CI) of diabetes across increasing quartiles of HbEO was 1.00 (reference), 1.45 (1.08, 1.96), 1.76 (1.31, 2.36), and 1.77 (1.22, 2.57), respectively (P-trend <0.001). Similar results were observed when analyses were stratified by smoking status, age, sex, race/ethnicity, and BMI. In a nationally representative sample of US adults, higher levels of HbEO were significantly associated with an increased prevalence of diabetes mellitus. Further prospective studies are needed to confirm these findings.