Context: Recent evidence suggests that Vitamin D may interact with the epigenome and play a role in the pain experience. In order for proper functioning to occur, there must be an adequate level of Vitamin D present, made possible by enzymatic reactions that allow Vitamin D to be biologically active. The purpose of this study was to explore the epigenetic landscape of genes involved in Vitamin D metabolism in individuals with and without chronic knee pain. Procedures: Community-dwelling individuals recruited as part of a larger study focused on knee pain provided demographic, clinical and pain-related information, as well as an intravenous blood sample to determine DNA methylation levels at CpG sites. Main Findings: There were differences in DNA methylation between those with and without pain in genes that code for enzymes related to Vitamin D metabolism: CYP24A1 (24-hydroxylase) and CYP27B1 (1--hydroxylase). There was also hypermethylation on the gene that codes for the Vitamin D receptor (VDR). Principal Conclusions: The presence of chronic pain is associated with epigenetic modifications in genes responsible for the expression of enzymes involved in Vitamin D metabolism and cellular function. These results lay groundwork in understanding the mechanism underlying the association between Vitamin D and chronic pain.