Our study aimed to identify differentially methylated regions (i.e., genomic region where multiple adjacent CpG sites show differential methylation) and their enriched genomic pathways associated with knee osteoarthritis pain (KOA). We recruited cognitively healthy middle to older aged (age 45-85) adults with (n = 182) and without (n = 31) self-reported KOA pain. We also extracted DNA from peripheral blood that was analyzed using MethylationEPIC arrays. The R package (Aryee et al., 2014) was used to perform methylation data preprocessing and quality control. To investigate biological pathways impacted by differential methylation, we performed pathway enrichment analysis using Ingenuity Pathway Analysis (IPA) to identify canonical pathways and upstream regulators. Annotated genes within ± 5 kb of the putative differentially methylated regions (DMRs, p < 0.05) were subjected to the IPA analysis. There was no significant difference in age, sex, study site between no pain and pain group (p > 0.05). Non-Hispanic black individuals were overrepresented in the pain group (p = 0.003). At raw p < 0.05 cutoff, we identified a total of 19,710 CpG probes, including 13,951 hypermethylated CpG probes, for which DNA methylation level was higher in the groups with highest pain grades. We also identified 5,759 hypomethylated CpG probes for which DNA methylation level was lower in the pain groups with higher pain grades. IPA revealed that pain-related DMRs were enriched across multiple pathways and upstream regulators. The top 10 canonical pathways were linked to cellular signaling processes related to immune responses (i.e., antigen presentation, PD-1, PD-L1 cancer immunotherapy, B cell development, IL-4 signaling, Th1 and Th2 activation pathway, and phagosome maturation). Moreover, in terms of upstream regulators, NDUFAF3 was the most significant (p = 8.6E-04) upstream regulator. Our findings support previous preliminary work suggesting the importance of epigenetic regulation of the immune system in knee pain and the need for future work to understand the epigenetic contributions to chronic pain.