Using the transcription activator-like effectors nucleases-mediated gene knockout technology, we have previously demonstrated that LH signaling is required for oocyte maturation and ovulation but is dispensable for testis development in zebrafish. Here, we have further established the fshb and fshr knockout zebrafish lines. In females, fshb mutant is subfertile, whereas fshr mutant is infertile. Folliculogenesis is partially affected in the fshb mutant but is completely arrested at the primary growth stage in the fshr mutant. In males, fshb and fshr mutant are fertile. The fertilization rate and histological structure of the testis is not affected. However, double knockout of fshb;lhb or fshr;lhr leads to all infertile male offspring. The key steroid hormones and steroidogenic genes are dramatically decreased in double knockout mutant (fshb;lhb and fshr;lhr) but not in single knockout mutant (fshb, lhb, fshr, and lhr) males. Furthermore, we have also demonstrated the constitutive activities of both FSH receptor (FSHR) and LH receptor in zebrafish and the compensatory role of LH by cross-reacting with FSHR in the fshb;lhr double mutant, thus explaining the phenotypic discrepancy observed among the ligand/receptor mutant lines. Taken together, our data established the following models on the roles of gonadotropin signaling in zebrafish gonad development. In females, FSH signaling is mainly responsible for promoting follicular growth, whereas LH signaling is mainly responsible for stimulating oocyte maturation and ovulation. In males, the functions of FSH and LH signaling overlap, and only disruption of both FSH and LH signaling could lead to the infertile phenotype. In the absence of FSH, LH could play a compensatory role by cross-reacting with FSHR in both male and female.