Abstract Background Single nucleotide polymorphisms (SNPs) of the gene encoding for human beta-defensin-1 (hBD1) are associated with increased risk of vertical HIV transmission (rs1799946) and reduced risk of periodontal disease (a predictor of poor obstetric outcome, rs1047031). There are conflicting reports from in-vitro studies on the effects of DEFB1 SNPs on protein expression. This study aimed to explore the association between the DEFB1 genotype and serum hBD1 expression in pregnant women. Methods In a retrospective, case-control study, genomic DNA and serum were extracted from blood collected from women at 11–13 weeks' gestation attending King's College Hospital between March, 2006, and September, 2010. The SNPs rs1799946 (5′UTR) and rs1047031 (3′UTR) were genotyped by Kompetitive Allele Specific PCR assay (LGC Genomics). Serum hBD1 concentration was measured by ELISA. Data were analysed with the Kruskal-Wallis and Mann-Whitney U tests. Findings DNA and serum samples were available for 292 women. Genotyping was successful in 286 women (98%, rs1047031) and 280 women (96%, rs1799946). Data were assessed initially in three groups (women who were homozygous for the ancestral allele, those who were heterozygous, and SNP homozygotes). SNP homozygotes (rs1047031) had a lower median serum hBD1 concentration than did women who were heterozygous or homozygous for the ancestral allele (3321 pg/mL [IQR 2874–5371] vs 5824 [4481–8338], p=0·0013). SNP homozygotes and heterozygotes (rs1799946) had a higher median serum hBD1 concentration than did women carrying only the ancestral allele (5955 [4608–8533] vs 5477 [4137–7088], p=0·0241). Interpretation This is the first in-vivo study, to our knowledge, showing that the DEFB1 SNPs rs1799946 and rs1047031 have a clear association with hBD1 protein expression. rs1799946 is associated with higher serum concentrations of hBD1 in the first trimester whereas rs1047031 genotype mediated lower serum concentrations of hBD1 in the first trimester. Funding Wellcome Trust, Wellbeing of Women.