Optimal peptide loading of MHC class I molecules is essential for antigen presentation to CD8+ Cytotoxic T lymphocytes. Endoplasmic Reticulum Aminopeptidase associated with Antigen Processing (ERAAP) is responsible for N-terminal trimming of peptides to the optimal length for stable loading and presentation on MHC class I. In humans, ERAAP is also known to play a role in pro-inflammatory cytokine receptor cleavage as well as regulating blood pressure and angiogenesis. Recently, a single nucleotide polymorphism (SNP) linkage analysis study has identified ERAAP as being associated with increased risk of the autoimmune inflammatory disorder, ankylosing spondylitis (AS). A HapMap comparison of AS positive patients against normal controls revealed susceptible and protective ERAAP alleles. In this study, SNP mutation of ERAAP is shown to alter the ability to trim peptides and facilitate IL-6R cleavage from the cell surface. Transfection of ERAAP-/- cells with individual SNP mutant hERAAP revealed a hierarchy of reduced function. Trimming function was further reduced when selected double SNP mutants were generated. Significantly, a mutant hERAAP, incorporating all the SNPs identified in the linkage analysis, completely abrogated its trimming function. The consistent reduction in activity of K528R and R725Q SNPs highlight these amino acids as important for ERAAP trimming function. Analysis of ERAAP alleles and haplotypes from AS patients identified novel polymorphic combinations which demonstrated a defective trimming activity in comparison to those identified in control samples. This has important implications on the role of these SNPs within ERAAP and the susceptibility of AS. Although the mechanism for the effect of SNP mutation on ERAAP function is unclear, it appears that they cause a dramatic effect on trimming of N-terminally extended peptides.