Avian uterine fluid (UF) has been demonstrated to prolong sperm survival, to maintain the fertility potential of the fowl sperm, and to impact the filling rate of sperm storage tubules (SST) from the utero-vaginal junction (UVJ). The purpose of this study was to identify UF metabolites related to long-term sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F-) sperm storage ability were used. Metabolites present in the UF either before or after insemination were identified and quantified using high resolution 1 H-NMR. The effect of the presence of sperm was investigated 24 hours, 1 week, 2 weeks and 3 weeks after a single artificial insemination. Thirteen metabolites were identified as discriminating between the UF collected before insemination from the two lines of hens. Among them, formate, organic acids (succinate, fumarate, lactate), dimethylamine and arabinose were significantly more abundant in F+ UF, whereas myo-inositol was significantly more abundant in F-UF (p<0.05). Pathway and metabolite set enrichment analyses revealed that preponderant metabolic pathways were over 2 representated in the F+ UF and involved pyruvate metabolism, citrate cycle and steroid biosynthesis (p<0.05). No metabolite was identified as discriminating in F+ UF, when compared before and after insemination. In contrast in the F-line of hens, 11 metabolites discriminated the UF collected before and after insemination. Actually, we observed that dimethylamine was significantly reduced, while fumarate and myo-inositol metabolites were significantly increased. Moreover, Warburg effect pathway involving glucose to lactate metabolism, was significantly enriched in F-after insemination. Our results indicate that in the hen, the metabolic composition of UF is related to the genetic traits F+/F-, and is differentially modified after insemination in both lines. It could be related to sperm storage. These lead us to conclude that: 1) the UF biochemical composition is associated with the sperm survival ability in SST, 2) is regulated by the presence of sperm, and 3) this regulation is related to the sperm survival ability in SST.