Objective To evaluate the contribution of the SPP1 rs11439060 and rs9138 polymorphisms, previously reported as autoimmune risk variants, in the RA genetic background according to anti-citrullinated protein antibodies (ACPAs) status of RA individuals. Methods We analyzed a total of 11,715 RA cases and 26,493 controls from 9 independent cohorts, all individuals were genotyped or had imputed genotypes for SPP1 rs11439060 and rs9138. The effect of the SPP1 rs11439060 and rs9138 risk-allele combination on OPN expression in macrophages and OPN serum levels was investigated. Results We provide evidence for a distinct contribution of SPP1 to RA susceptibility according to ACPA status: the combination of ≥ 3 SPP1 rs11439060 and rs9138 common alleles was associated mainly with ACPA negativity (P = 1.29x10-5, ORACPA-negative 1.257 1.135–1.394) and less with ACPA positivity (P = 0.0148, ORACPA-positive 1.072 1.014–1.134]). The odds ratios between these subgroups (i.e., ACPA-positive and -negative) significantly differed (P = 7.33x10-3). Expression quantitative trait locus analysis revealed an association of the SPP1 risk-allele combination with decreased SPP1 expression in peripheral macrophages from 599 individuals. To corroborate these findings, we found an association of the SPP1 risk-allele combination and low serum level of secreted OPN (P = 0.0157) as well as serum level of secreted OPN correlated positively with ACPA production (P = 0.005; r = 0.483). Conclusion We demonstrate a significant contribution of the combination of SPP1 rs11439060 and rs9138 frequent alleles to risk of RA, the magnitude of the association greater in patients negative for ACPAs.