ObjectiveThe osteoarthritis (OA)–associated single‐nucleotide polymorphism (SNP) rs11583641 is located in COLGALT2, encoding a posttranslational modifier of collagen. In cartilage, the SNP genotype correlates with DNA methylation in a putative enhancer. This study was undertaken to characterize the mechanistic relationship between rs11583641, the putative enhancer, and COLGALT2 expression using cartilage samples from human patients and a chondrocyte cell model.MethodsNucleic acids were extracted from articular cartilage samples obtained from patients with OA (n = 137). Samples were genotyped, and DNA methylation was quantified at 12 CpGs using pyrosequencing. The putative enhancer was deleted in Tc28a2 chondrocytes using clustered regularly interspaced short palindromic repeat/Cas9, and the impact on nearby gene expression was determined using real‐time quantitative polymerase chain reaction. Targeted modulation of the epigenome using catalytically dead Cas9 (dCas9) constructs fused to DNA methyltransferase 3a or ten–eleven translocase 1 allowed for the investigation of a causal relationship between DNA methylation and enhancer activity.ResultsThe genotype at rs11583641 correlated with DNA methylation at 3 CpGs, and the presence of the OA risk allele, C, corresponded to reduced levels of methylation. Deletion of the enhancer resulted in a 2.7‐fold reduction in COLGALT2 expression. Targeted methylation and demethylation of the CpGs had antagonistic effects on COLGALT2 expression. An allelic imbalance in the expression of COLGALT2 was identified in the cartilage from patients with OA, with relative overexpression of the OA risk allele. Allelic expression ratios correlated with DNA methylation at 4 CpGs.ConclusionCOLGALT2 is a target of OA genetic risk at this locus. The genotype at rs11583641 impacts DNA methylation in a gene enhancer, which, in turn, modulates COLGALT2 expression. COLGALT2 encodes an enzyme that initiates posttranslational glycosylation of collagens and is therefore a compelling OA susceptibility target.