AbstractRecent studies have suggested that epigenetic alterations, particularly DNA methylation, play a crucial role in the pathogenesis of insulin resistance. Folate is a key source of the one-carbon group for DNA methylation, whereas the association and mechanistic linkage between folate status and insulin resistance remains unclear with very limited experimental support. We performed a cross-sectional study of 1530 nondiabetic adults in the 2011–2012 National Health and Nutrition Examination Survey (NHANES). We examined associations between serum folate and insulin resistance using multiple linear regression models adjusted for potential confounders. We detected a significant inverse relationship for serum folate, where a 25% increase in serum folate was associated with a 3.06% (95% CI, −4.72, −1.37) and 2.77% (95% CI, −4.36, −1.77) decline in HOMA-IR and insulin respectively, and a 2.55% (95% CI, 0.93, 4.21) increase in G/I ratio. Our findings demonstrate that serum folate was inversely associated with insulin resistance in U.S. nondiabetic adults.