The IGH V3-23*01 gene is used in the formation of the canonical combining site which dominates the human antibody repertoire to the Haemophilus influenzae type b (Hib) polysaccharide (PS). An allele of the human IGH V3-23*01 gene, known as V3-23*03, differs from V3-23*01 in nine bases, eight of which are located in the second complementarity determining region. These eight differences encode five amino acid substitutions. In this study we investigated whether the V3-23*03 sequence polymorphism affected Hib PS binding. We constructed two Fab fragments that had the canonical Hib PS combining site VH-VL configuration but that had either V3-23*01 or V3-23*03. Radioantigen binding assay showed that on a concentration basis the V3-23*03 Fab was 20-fold more effective in binding Hib PS than the V3-23*01 Fab. The V3-23*03 Fab was 4-fold more effective than the V3-23*01 Fab in mediating facilitated bactericidal activity against Hib organisms. These findings identify a functional consequence of V3-23 allelism, and suggest that utilization of the V3-23*03 gene in the human Hib PS repertoire would generate canonical antibodies with higher affinity and protective efficacy than canonical antibodies utilizing V3-23*01. Thus, IGH V gene allelic variation has the potential to impact the generation of protective immunity to Hib.