The RXFP2 gene is a key regulator of horn morphology in sheep ( Ovis aries Linnaeus), with a 1.78-kb insertion in its 3′-untranslated region (UTR) previously linked to the polled phenotype. However, horned individuals homozygous for this insertion (1.78 kb +/ +) have been observed in typically polled breeds such as Hu sheep, suggesting additional regulatory mechanisms. In this study, a significant horn-associated quantitative trait locus (QTL) on chromosome 10 was identified through genome-wide association analysis which was consistent with previous study. We found that the 1.78-kb insertion in the 3′-UTR of RXFP2 is associated with horn status across sheep breeds, and that elevated RXFP2 expression correlates with horn development even in 1.78 kb +/ + individuals, as revealed by expression analyses in multiple breeds. RNA-seq and hormonal profiling further demonstrated that the INSL3/RXFP2 signaling axis, particularly its interactions with hormones such as testosterone, plays a central role in horn morphogenesis. These results indicate that horn development is modulated not only by structural variations in RXFP2, but also through its transcriptional and hormonal regulation, providing new insights into the polygenic basis of horn formation and informing breeding strategies for polled sheep.