Simple SummaryTheR-spondin 1(RSPO1) gene is considered to play an important role in the gonadal development of female vertebrates, and estrogen plays an important regulatory role in the gonadal development of female chicken embryos. However, the expression ofRSPO1in the gonad of chicken embryos and the relationship betweenRSPO1expression and estrogen level are not fully understood. In this study, we found thatRSPO1showed female-biased expression in chicken embryonic gonads, but there was no significant difference in the expression ofRSPO1between female and male gonadal cells. Moreover, the expression ofRSPO1could be regulated by the alteration of estrogen levels at both cellular and in vivo levels, suggesting that the female-biased expression ofRSPO1in the gonads of chicken embryos is estrogen-dependent. This study will provide a theoretical basis for studying the molecular mechanism ofRSPO1in the development of chicken embryo ovary.AbstractThe mechanism of sex determination in chickens, especially the molecular mechanism of female ovarian development, has not yet been fully elucidated. Previous studies have shown thatRSPO1, which is associated with ovarian development in mammals, might have a conserved role in chickens. In this study, we systematically investigated the spatiotemporal expression pattern ofRSPO1in various tissues, especially gonads, of male and female chicken embryos using qPCR and Western blotting, and we explored its correlation with the expression of key genes in the estrogen pathway using drug treatment or gene overexpression in vivo and in vitro. Our results reveal thatRSPO1was widely expressed in all examined tissues of chicken embryos, showing a female bias in gonadal tissues at both the mRNA and protein levels. Surprisingly,RSPO1was not differentially expressed between male and female gonadal cells with fadrozole-induced estrogen pathway blockades, and furthermore, estradiol-induced estrogen stimulation altered the expression ofRSPO1. In addition, overexpression ofRSPO1in gonadal cells induced the mRNA expression of its downstream target genes,Wnt family member 4(WNT4) andCatenin beta 1(CTNNB1), and that ofestrogen receptor α(ERα), an estrogen pathway gene. In summary, this study provided new evidence for elucidating the role ofRSPO1in ovarian development in poultry.