Matrix Metalloproteinase-9 (MMP9) degrades the Extracellular Matrix (ECM), participates in mammary gland remodeling, and inhibits mammary epithelial cell apoptosis in goats. To investigate the transcriptional regulatory mechanism of the MMP9 promoter region, we analyzed the expression pattern of MMP9 in dairy goats by qRT‒PCR and cloned the promoter region by PCR. Deletion analysis indicated that the MMP9 gene core promoter region was located upstream of the transcription start site in the-715 bp to-926 bp region. We predicted three Specificity protein 1 (Sp1) binding sites in the MMP9 core promoter region, and performed targeted mutations on these three sites. The c. 1863 G> A mutation in the MMP9 gene increased the promoter transcriptional activity and may be associated with an additional Serum Response Factor (SRF) transcription factor-binding site. Association analysis revealed that c. 1863 G> A was significantly associated with milk fat percentage in dairy goats, which was significantly higher in goats with the AG genotype (4. 71±0. 02%) than in goats with the GG genotype (4. 61±0. 05%). This study lays a foundation for subsequent analysis of the transcriptional regulatory mechanism of MMP9 and exploration of its biological functions.