A high level of androstenone in porcine adipose tissue is a major factor contributing to boar taint. Porcine hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (3β-HSD, also known as HSD3B1) plays a key role in the hepatic metabolism that catalyzes androstenone to β-androstenol. Therefore, 3β-HSD is a candidate gene for boar taint. This study aimed to investigate functional 3β-HSD polymorphisms in Duroc pigs. We found eight single nucleotide polymorphisms (SNPs) in the full-length porcine 3β-HSD. Four of the SNPs had restriction enzyme sites, and we genotyped them in 147 uncastrated male Duroc pigs using a polymerase chain reaction-restriction fragment length polymorphism method. Pigs with the GG genotype at the g.165262G>A locus (SNP5) had significantly lower androstenone levels than did those with other genotypes (P = 0.030). SNP5 also was associated with differences in 3β-HSD mRNA levels: pigs with the GG genotype had higher levels than those with other genotypes (P = 0.019). The SNP5 polymorphism could affect the hepatic catabolism of androstenone and consequently impact androstenone accumulation in the adipose tissue. Therefore, SNP5 in the 3β-HSD of Duroc pigs could be a useful selective marker for decreasing boar taint.