SPAN-Xb is a novel cancer-testis antigen in multiple myeloma. We recently demonstrated that SPAN-Xb expression in myeloma cells is regulated through promoter methylation and could be upregulated by IL-7 and GM-CSF. In this present study, we set out to investigate the mechanism of SPAN-XB expression and the promoter association with the methyl-CpG binding protein (MeCP2). Elucidation of these interactions is likely shed light on potential therapeutic strategies to upregulate antigen levels for SPAN-Xb-based tumor vaccines. Using a panel of truncated promoter constructs, we localize the core sequence of SPAN-XB promoter to the 73 bp at the 3′ end of the promoter, a region within the full length promoter that lacks CpG dinucleotides. Reporter gene expression assays showed that the core promoter function is significantly modulated by the adjacent CpG sequences. Chromatin immunoprecipitation assays revealed a specific association of MeCP2 with the promoter, and MeCP2 binding strongly correlated with repression of SPAN-XB gene. Reactivation of the SPAN-XB gene by 5-azacytidine treatment resulted in the loss of MeCP2 from this site. We, therefore, conclude that SPAN-XB core promoter function in myeloma cells is associated with MeCP2 protein binding and regulated by specific CpG dinucleotide sequences. © 2006 Wiley-Liss, Inc.