Organic arsenic intake from seafood is one of the major arsenic exposure routes among the general population. However, organic arsenic metabolism in the human body is not yet clear. The goal of this study was to explore the effects of genetic polymorphisms of human PNP, As3MT and GSTO1 on organic arsenic metabolism among study subjects after oyster ingestion. During the one-week dietary controlled study, fifty study subjects were provided all their daily meals without seafood, except for two designated amounts of oyster given on the fourth day. First morning voided urine samples were provided by the study subjects for 7 consecutive days and analyzed with HPLC-ICP-MS for As3+, As5+, monomethylarsonic acid, and dimethylarsinic acid (DMA). Blood samples were collected later for genetic polymorphisms analysis of PNP, As3MT and GSTO1. Study subjects were categorized into “fast-” (n = 32), “medium-” (n = 13) and “slow-metabolizing” (n = 5) groups based on the number of days after ingestion needed for each subject's urinary DMA level reaching peak. Allele frequencies of single nucleotide polymorphisms (SNP) in intron 6 (G/C, p = 0.024) and in intron 10 (T/C, p = 0.039) of As3MT were significantly associated with the urinary DMA excretion. General estimating equation model analysis indicated that the variants of SNP (G>C) in intron 6 and SNP (T > C) in intron 10 of As3MT were respectively associated with higher or lower urinary DMA level by approximately 9 μg L−1. As3MT was suggested to be one of the major factors affecting the metabolism of dietary organic arsenic in terms of urinary DMA level.