BACKGROUND: Although relationships between smoking/high cotinine and type 2 diabetes have consistently been observed, few studies have investigated the relationship between cotinine and underlying pathophysiological defects that characterize diabetes aetiology. This study aimed to test the associations between cotinine and measures of insulin resistance or insulin secretion. METHODS: This analysis included 5,751 non-diabetic adult American from the National Health and Nutrition Examination Survey (NHANES) from 2007-2012. Insulin function was represented with two indexes: insulin resistance index (HOMA-IR) and insulin secretion index (HOMA-B) estimated by homeostasis model assessment. We categorized cotinine levels into quartiles and estimated the odds of HOMA-IR in the 4th quartile and HOMA-B in the 1st quartile among cotinine categories using multiple logistic regression models. RESULTS: Cotinine concentration was not associated with the risk of high HOMA-IR. Association of cotinine with low HOMA-B existed and differed by race/ethnicity (P for interaction<0.05). High cotinine concentration (in the 4th quartile) was associated with an increased risk of low HOMA-B compared with low cotinine concentrations(1st -2nd quartiles) among white (odds ratio[OR], 1.51 [95% confidence interval[CI], 1.16-1.97]) or black participants (OR, 2.98 [95%CI, 1.90-4.69]) but not among Mexican (OR, 1.79 [95%CI, 0.90-3.53]) or other Hispanic(OR, 1.02 [95%CI, 0.56-1.86]) participants. Such associations remained significant even after further adjustment for HOMA-IR. CONCLUSIONS: High cotinine is associated with decreased insulin secretion function only in white and black non-diabetic U.S. adult population. Results evaluating cotinine in ethnically homogeneous populations may not be broadly generalizable to other racial/ethnic groups.