We previously demonstrated that the Alzheimer's disease (AD) associated risk allele, rs3865444^C, results in a higher surface density of CD33 on monocytes. Here, we find alternative splicing of exon 2 to be the primary mechanism of the genetically driven differential expression of CD33 protein. We report that the risk allele, rs3865444^C, is associated with greater cell surface expression of CD33 in both subjects of European and African–American ancestry and that there is a single haplotype influencing CD33 surface expression. A meta-analysis of the two populations narrowed the number of significant SNPs in high linkage disequilibrium (LD) (r² > 0.8) with rs3865444 to just five putative causal variants associated with increased protein expression. Using gene expression data from flow-sorted CD14⁺CD16⁻ monocytes from 398 healthy subjects of three populations, we show that the rs3865444^C risk allele is strongly associated with greater expression of CD33 exon 2 (p_META = 2.36 × 10⁻⁶⁰). Western blotting confirms increased protein expression of the full-length CD33 isoform containing exon 2 relative to the rs3865444^C allele (P < 0.0001). Of the variants in strong LD with rs3865444, rs12459419, which is located in a putative SRSF2 splice site of exon 2, is the most likely candidate to mediate the altered alternative splicing of CD33's Immunoglobulin V-set domain 2 and ultimately influence AD susceptibility.