GTP cyclohydrolase 1 (GCH1) is the initial and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, which is an essential cofactor for biosynthetic enzymes of dopamine, serotonin, and nitric oxide. In the present study, the association of functional polymorphism of the GCH1 gene (C+243T, rs841) with personality traits was examined in 902 healthy Japanese subjects. Personality traits were assessed by the Temperament and Character Inventory (TCI), and the GCH1 genotype was detected by a PCR–RFLP method. There were no significant main effects of the GCH1 genotype on the seven TCI dimension scores, but significant interaction effects between the GCH1 genotype and gender were found on the scores of novelty seeking. Post-hoc analysis revealed that males with the C/C genotype had higher scores of novelty seeking than those with the C/T genotype or those with the T/T genotype, while in females the scores of novelty seeking were not different among the genotype groups. The present study thus suggests that the C+243T polymorphism of the GCH1 gene affects the personality trait of novelty seeking in males.