OBJECTIVE: This study aimed to refine the interaction between cigarette smoking and human leukocyte antigen (HLA) polymorphisms in seropositive rheumatoid arthritis (RA), in the context of a recent amino-acid based HLA model for RA susceptibility. METHODS: We imputed HLA amino acids and classical alleles from case-control Immunochip array data of 3,588 Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA; case/control=1654/1934), 589 Nurses' Health Study (NHS; 229/360) and 2,125 Korean (1390/735) subjects. We examined interaction effects between heavy smoking (>10 pack-years) and genetic risk score (GRS) from RA-associated amino-acid positions (11, 13, 71 and 74 in HLA-DRβ1; 9 in HLA-B; and 9 in HLA-DPβ1) and from HLA-DRβ1 four-amino-acid haplotypes with an attributable proportion due to interaction (AP) using the additive interaction model. RESULTS: Heavy smoking and all investigated HLA amino-acid positions and haplotypes were associated with RA susceptibility in all three populations. In the interaction analysis, we found a significant deviation from the expected additive joint effect between heavy smoking and the HLA-DRβ1 amino-acid haplotype in all three studies (0.416≤AP≤0.796). We further identified the key interacting variants as being located at amino-acid positions 11 and 13 of HLA-DRβ1 but not the other RA-risk amino-acid positions in all populations. At the positions 11 and 13, there were similar patterns between RA-risk effects and interaction effects of residues. CONCLUSION: Our findings of significant gene-environment interaction effects implicate that a physical interaction between citrullinated auto-antigens produced by smoking and HLA-DR molecules is characterized by the HLA-DRβ1 four-amino-acid haplotype, primarily by the positions 11 and 13. This article is protected by copyright. All rights reserved.