To explore the role of inducible nitric oxide synthase (Nos2) in fish growth regulation, we generated nos2a deficient (nos2a^-/-) zebrafish using CRISPR/Cas9 technology for the first time. By monitoring their growth process,we found the growth trend of nos2a^-/-zebrafish was slower than nos2a^+/+zebrafish, and the adult body size of nos2a^-/-zebrafish was smaller than that of nos2a^+/+zebrafish (P<0.01):the mean length and weight of nos2a^+/+zebrafish were 29.53 mm and 512.8 mg, respectively,that of nos2a^-/-zebrafish was 26.34 mm and 394.4 mg. Additionally, there was no notable difference in the density of muscle fibers (P=0.718) and sectional area of vertebrae (P=0.548)between nos2a^+/+ and nos2a^-/-zebrafish. Moreover,compared with nos2a^+/+zebrafish at 8 dph (days post hatching), the mRNA level of growth hormone (gh1, P<0.01) and insulin-like growth factor-1 (igf1, P<0.001) of nos2a^-/-zebrafish at 8 dph were lower significantly; and it was the same as that at 68 dph:gh1, P<0.000 1; igf1, P<0.000 1. These findings indicate that the defect of nos2a negatively affected zebrafish growth, and the reduced gene mRNA level of growth hormone was the potential factor.