Zebrafish (Danio rerio) were used as a model fish, and the technique of RNA interference (RNAi) was employed to knockdown three subunits of the gonadotropin alpha (GtHα, common α), follicle-stimulating hormone beta (FSHβ), and luteinizing hormone beta (LHβ) genes. Three short-hairpin RNA (shRNA) expression vectors and three mismatched shRNA expression vectors as controls for each subunit gene were constructed, and the depression efficiency was tested in vivo by microinjection; the RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and green fluorescent protein (GFP) analyses. Expression of GtH mRNA was obviously and more efficiently depressed by GtHα RNAi expression compared with the other two subunits. A GtHα morpholino analysis showed that the GtHα morpholino led to suppression of embryonic development and the production of embryonic mutants as a result of an injection of GtHα -shRNA. Taken together, these results show that GtHα-shRNA, which more efficiently targets RNAi, may have an essential role in the further development of sterility technology of transgenic fish for biosafety purposes.