Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components for acceptance of sperm in SST, sperm functions maintenance during weeks, sperm release out of SST, and their ascending through the uterus. The study of UF and SST is essential to better understand sperm storage process. The objectives were to identify proteins modulated in the hen’s oviduct by sperm, and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F-) sperm storage ability were used. GeLC-MS/MS strategy was used to establish a quantitative inventory of proteins regulated after insemination in both lines. Consequently the identification of high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers was investigated in uterovaginal junction by immunohistochemistry. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content, as well as in the SST epithelium. In F+ hens, the ANXA4 protein mobilization in apical part of SST cells after insemination was associated to increased levels of some proteoglycans and binding proteins, as well as antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F- hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity, and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system is major to regulate sperm storage.