Because of prolonged sperm storage in their oviduct, domestic hens can produce fertile eggs for up to 3 weeks following a single artificial insemination (AI). The oviduct secretions may have an impact on sperm survival but its composition during fertilization is unknown.  In the present study, we compared the proteomic content of uterine fluid collected from two distinct lines of hens. The first displays a shorter period of sperm storage (10 days, line DPF-) whereas the second displays a longer period of sperm storage (21 days, DPF+). The aim was to identify proteins or peptides that may be involved in spermatozoa survival. Uterine fluid was collected 10h after oviposition into a plastic tube placed at the entrance of the everted vagina, either before and 24h after an artificial insemination. Bottom up approach using SDS-PAGE and nano LC-MS/MS was performed. Data were matched against NCBInr database (2014) using Mascot and identifications were validated by the peptide and protein Prophet algorithm using Scaffold software. To determine the differences in protein expression, spectral counting and XIC quantitative methods were employed using Scaffold Q+ (p<0.05, ratio >2). MS quantifications were validated by western blot for few proteins. A total of 41 and 53 proteins were differentially expressed in DPF- and DPF+ fluid, respectively (p<0.05) in response to AI. Moreover, three proteins were only present in DPF- line, whereas 16 proteins were only present in DPF+ uterine fluids. In conclusion, the presence of sperm in genital tract induced quantitative differences of the protein content of the uterine fluid, in DPF- and in DPF+ lines.