Prostacyclin synthase (CYP8A1) is an enzyme responsible for the biosynthesis of prostacyclin (PGI₂) which inhibits platelet activation and exhibits anti-inflammatory effect. The objectives of this study were to identify CYP8A1 genetic variants and characterize functional consequences of CYP8A1 variants. In total, 27 variants including four previously unidentified single-nucleotide polymorphisms (SNPs) were identified by direct DNA sequencing in Koreans (n = 48). Among them, CYP8A1 A447T and E314Stop were newly assigned as CYP8A1^∗5 and CYP8A1^∗6 by the Human Cytochrome P450 Allele Nomenclature Committee, respectively. CYP8A1^∗5 was found in the heme binding area in three individuals as a heterozygous mutation. To investigate the functional change of CYP8A1^∗5, CYP8A1^∗5 and wild-type CYP8A1 protein were overexpressed in an Escherichia coli expression system and purified. Metabolism of PGH₂ by the CYP8A1^∗5 protein exhibited significantly decreased activity, resulting in a 45% decrease in V_max and a 1.8-fold decrease in intrinsic clearance compared to the wild-type. Based on the predicted crystal structure of CYP8A1^∗5 using the Molecular Operating Environment platform, the distance from CYP8A1 Cys441 to the heme was altered with a significantly changed binding free energy for the mutant protein. Further studies would be needed to determine the effect of CYP8A1^∗5 on PGI₂ levels in humans.