In the zebrafish, no sex-determining gene has been identified, while some sex-related genes, suchas cyp19a1a and amh, show sexually dimorphic expression. Interestingly, most of these genes areexpressed in the somatic cells.With increasing evidence suggesting roles of germ cells in gonadaldifferentiation, there is an increasing interest in the factors released by the germ cells for thebidirectional communication between the two compartments. We have reported that Gdf9/gdf9 isan oocyte-specific factor in the zebrafish, similar to that of mammals. Whether and how Gdf9 isinvolved in gonadal differentiation is unknown. In this study, we compared the expression levels ofgdf9, cyp19a1a, and amh among several other sex-related genes in the gonads before, during, andafter sex differentiation. The expression of gdf9 started in the gonads before sex differentiation, andits level surged in the differentiated ovary. Its expression pattern was similar to that of cyp19a1a,but reciprocal to amh expression. Using recombinant zebrafish Gdf9 (rzfGdf9), we further showedthat Gdf9 significantly suppressed the expression of amh while increased that of activin betasubunits (inhbaa and inhbb) in vitro. Although gdf9 and cyp19a1a showed co-expression duringgonadal differentiation, we only observed a slight but not significant response of cyp19a1a torzfGdf9. Knocking down the expression of gdf9 and cyp19a1a with vivo-morpholinos caused amale-skewed sex ratio. Our data suggested that Gdf9 is likely involved in promoting oocyte/ovary differentiation in the zebrafish and it may act by suppressing amh expression, at least partly, in thesomatic cells. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved.