Steroidogenic factor 1 (sf1) is an important regulator of gonad development and function in mammals. However, study of sf1 in fish is limited to cloning and expression and in vitro experiments. Using antisense RNA we knockout transcription of the sf1 gene in Nile tilapia Oreochromis niloticus, and obtain experimental fish in vivo. We demonstrate that antisense RNA can silence sf1 transcription and protein expression, and report suppression of sf1 transcription to affect gonad development and external genitalia formation in Nile tilapia. We also report disfunction of retinal metabolism and fatty acid metabolism to be important causes of weight gain and gonad abnormality with sf1 suppression. The feasibility of using antisense RNA for gene editing in fish is verified, and a new way of studying gene function and performing biological breeding is presented.