The origin of teleost skin color is from chromatophores. Different chromatophores synthesize distinct pigments, including melanin, carotenoid, pteridine and purine. Sepiapterin is a yellow pteridines, which could be transformed into tetrahydrobiopterin (BH₄) and yellow/red pteridines pigments together with sepiapterin reductase (SPR), dihydrofolate reductase (DHFR) and other enzymes. SPR is the last enzyme in the process of de novo BH4 synthesis. In order to explore the function of spr in koi carp color formation, this study amplified the whole cDNA of spr and analyzed the spatio-temporal profile. Furthermore, we also detected the expression and distribution of SPR in the skin, fins and scales of koi carp with different colors by Western blot and immunochemistry methods. The results showed that the size of spr cDNA was 879 bp, including 132 bp and 134 bp 5' and 3' untranslated regions, and a 510 bp open reading frame encoding 170 amino acids. Sequences alignment and phylogenetic analysis revealed the spr gene of koi carp contained a adh_short_C2 conserved domain and had 97.65% similarity with gold fish. spr was expressed in every tissue, especially highest expressed in skin. In the four ontogenetic stages, the expression level of spr firstly decreased, then rose. The expression level of spr mRNA and protein in skins, fins and scales displayed the same condition among three colors (whole red koi carp, whole white koi carp and kohaku koi carp). The highest expression level was detected in whole red koi carp and rarely in white skin, fins and scales of kohaku koi carp. The immunohistochemical positive signals were detected in both skins of whole red and whole white koi carp, and intensively exhibited in red skin compared with white skin. All the results might indicate that the spr gene has relationship with the xanthophores/erythrophores differentiation and formation, involved in koi carp color formation.