Objective: To explore the association between insulin resistance (IR) and genome-wide DNA methylation based on Shanghai twin study. Methods: Monozygotic twins (MZ) from Shanghai were recruited during 2012-2013, 2017-2018, and 2022-2023. Data were collected by questionnaire survey, physical examination and laboratory tests. Genome-wide DNA methylation was quantified. Generalized linear mixed effect model was applied to analyze the association between methylation level at each site and homeostatic model assessment 2-insulin resistance (HOMA2-IR). Non-paired and paired designs were used to assess the association between DNA methylation and phenotype of IR. Cluster analysis was conducted to identify the clusters of top significant sites. Generalized linear regression was performed to examine the differential methylation patterns from clusters. Results: A total of 100 MZ pairs were included in this study. Hypermethylated cg10535199-2q23.1 ( β =0.74%, P =1.51×10 -7 , OR =1.06, 95% CI : 1.03-1.09) and ch.17.49619327- SPOP ( β =0.23%, P =7.54×10 -7 , OR =1.17, 95% CI : 1.08-1.28) were identified with suggestive significance. After correcting for multiple testing, no sites reached genome-wide significance. There was no statistical significance in the paired analysis. Two clusters with hypomethylated ( β =-0.39%, P <0.001) and hypermethylated ( β =0.47%, P <0.001) patterns were observed for HOMA2-IR. Conclusions: IR was significantly associated with DNA methylation, and genetic factors might contribute to the association.