Recent 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.