This study characterized the human apolipoprotein H [APOH; β2‐glycoprotein I (β2GPI)] promoter and its variants by in vitro functional experiments and investigated their relationship with human plasma β2GPI levels. We examined the individual effects of 12 APOH promoter single nucleotide polymorphisms in the 5′ flanking region of APOH (∼ 1.4 kb) on luciferase activity in COS‐1 cells and HepG2 cells and their impact on plasma β2GPI levels in 799 American White people, the DNA binding properties of the APOH promoter using an electrophoretic mobility shift assay in HepG2 cells, the effects of serial deletion analysis of the APOH 5′ flanking region in COS‐1 and HepG2 cells and cross‐species conservation of the APOH promoter sequence. The variant alleles of three single nucleotide polymorphisms (−1219G>A, −643T>C and −32C>A) showed significantly lower luciferase expression (51, 40 and 37%, respectively) as compared with the wild‐type allele. The electrophoretic mobility shift assay demonstrated that these three variants specifically bind with protein(s) from HepG2 cell nuclear extracts. Three‐site haplotype analysis (−1219G>A, −643T>C and −32C>A) revealed one haplotype carrying −32A (allele frequency = 0.075) to be significantly associated with decreased plasma β2GPI levels (P < 0.001). Deletion analysis localized the core APOH promoter to ∼ 160 bp upstream of ATG codon with the presence of critical cis‐acting elements between −166 and −65. Cross‐species conservation analysis of the APOH promoters of seven species indicated that basic promoter elements are highly conserved across species. In conclusion, we have characterized the functional promoter of APOH and identified functional variants that affect the transcriptional activity of the APOH promoter.