The common prothrombin gene cleavage site mutation 20210G>A is associated with elevated prothrombin levels and thrombosis. The pathomechanism of the 20210G>A mutation was explained by increased mRNA formation and/or more efficient translation. Human studies also showed an influence of the intronic 19911A>G polymorphism on prothrombin activity. We established HepG2 cell lines stably transfected with prothrombin mini-genes containing the last 2 prothrombin exons, the last intron, 3' untranslated region (UTR), and flanking sequence. The highest mRNA expression and protein activity resulted from the mutant haplotype 19911A-20210A. Haplotypes with wild-type cleavage site (19911A-20210G, 19911G-20210G) also differed significantly as a consequence of the intronic 19911 mutation; the 19911G-20210G haplotype showed lower expression than the 19911A-20210G haplotype, whereas previous clinical studies have reported elevated prothrombin activity with the 19911G-20210G haplotype. The cleavage site pattern was homogeneous with 20210A, which may cause a favorable intracellular processing, and heterogeneous with 20210G. In an independent assay for splicing efficiency, 19911G showed about 30% higher efficiency than 19911A. We conclude that the intronic 19911A>G single nucleotide polymorphism is itself functional and changes splicing efficiency by altering a known functional pentamer motif. Further studies are needed to define the value of additional prothrombin 19911 genotyping for thrombophilia screening, especially in cases heterozygous for 20210G>A.