The B cell inhibitory receptor FcγRIIB plays crucial roles in the maintenance of self-tolerance. We have identified a polymorphism FCGR2B c.695T>C that results in the non-conservative replacement of 232Ile at the transmembrane helix to Thr and demonstrated the association of the polymorphism with susceptibility to systemic lupus erythematosus (SLE) in Asians. In this study, we examined the impact of FCGR2B c.695T>C on the functional properties of FcγRIIB by expressing each allele product in a human B cell line ST486 lacking endogenous FcγRIIB. FcγRIIB 232Thr was found to be significantly less potent than wild-type 232Ile in inhibiting B cell receptor (BCR)-mediated phosphatidylinositol-3,4,5-trisphosphate accumulation, Akt and PLCγ2 activation and calcium mobilization, and to display decreased levels of tyrosine phosphorylation and SH2-containing 5′-inositolphosphate phosphatase recruitment compared with 232Ile after IgG Fc-mediated coligation with BCR. Notably, a quantitative analysis of the subcellular distribution of FcγRIIB using ¹²⁵I-labeled anti-FcγRIIB revealed that FcγRIIB 232Thr is less effectively distributed to detergent-insoluble lipid rafts than 232Ile, findings in accordance with the importance of the transmembrane amino acid residues, in particular large hydrophobic amino acids including Ile, in the association of membrane proteins with lipid rafts. Given the crucial roles of lipid rafts in integrating BCR signaling, decreased association of FcγRIIB 232Thr could contribute to its impaired inhibitory potential. Collectively, the present findings indicate that the Ile232Thr substitution affects the localization and function of FcγRIIB and that the molecular mechanism may link the polymorphism and susceptibility to SLE.