LY6K is associated with development of the immune response and carcinogenesis. Elevated LY6K by AP-1 activation induces invasion and metastasis by activating the ERK signaling pathway. However, the exact epigenetic mechanism of LY6K gene expression has not been clarified. The CpG island and 5′CGI shore in the region around the transcription start site of the LY6K were predicted using the UCSC genome browser and Methprimer software to elucidate the epigenetic mechanism of LY6K gene regulation and expression. We performed the methylation-specific PCR, bisulfite pyro-sequencing, and bisulfite sequencing to investigate the DNA methylation status of CpG sites from −400 to +500 on the LY6K CpG island and 5′CGI shore. ChIP and ChIP-MSP analysis were performed to confirm the histone modification and DNA methylation status. To validate the inverse correlation between LY6K 5′CGI shore methylation and LY6K expression in vivo, we performed bisulfite pyro-sequencing and tissue microarray using breast carcinoma samples. LY6K expression was inversely correlated with methylation status of CGI and 5′CGI shore in the LY6K of human breast cancer. Moreover, DNA methylation status of LY6K was correlated with histone modification. Treatment of 5-Aza-dC with low LY6K expression caused elevated LY6K expression leading to wound healing. An understanding of epigenetic changes in LY6K may contribute to diagnosing carcinogenic risk and to predict the outcome of patients with breast cancer. Citation Format: Hyun Kyung Kong, Sae Jeong Park, Ye Sol Kim, Jong Hoon Park. Epigenetic modification of LY6K in CGI shore and CGI regulates LY6K gene activation and metastatic function in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4771. doi:10.1158/1538-7445.AM2015-4771