Background The link between DNA methylation, obesity, and adiposity-related diseases in the general population remains uncertain. Methods and Findings We conducted an association study of body mass index (BMI) and differential methylation for over 400,000 CpGs assayed by microarray in whole-blood-derived DNA from 3,743 participants in the Framingham Heart Study and the Lothian Birth Cohorts, with independent replication in three external cohorts of 4,055 participants. We examined variations in whole blood gene expression and conducted Mendelian randomization analyses to investigate the functional and clinical relevance of the findings. We identified novel and previously reported BMI-related differential methylation at 83 CpGs that replicated across cohorts; BMI-related differential methylation was associated with concurrent changes in the expression of genes in lipid metabolism pathways. Genetic instrumental variable analysis of alterations in methylation at one of the 83 replicated CpGs, cg11024682 (intronic to sterol regulatory element binding transcription factor 1 [SREBF1]), demonstrated links to BMI, adiposity-related traits, and coronary artery disease. Independent genetic instruments for expression of SREBF1 supported the findings linking methylation to adiposity and cardiometabolic disease. Methylation at a substantial proportion (16 of 83) of the identified loci was found to be secondary to differences in BMI. However, the cross-sectional nature of the data limits definitive causal determination. Conclusions We present robust associations of BMI with differential DNA methylation at numerous loci in blood cells. BMI-related DNA methylation and gene expression provide mechanistic insights into the relationship between DNA methylation, obesity, and adiposity-related diseases.