Abstract: Nitrogen is indispensable for plant growth and development. We can increase yield in many crops using nitrogen application, however, excessive application of nitrogen fertilizer is uneconomic and harmful to the environment. Forages generally suffer more severe nutrition stress than other crops. In this study, metabolites were analyzed with LC-ESI-MS (liquid chromatography-electrospray ionization-mass spectrometry) in tall fescue leaves under normal and low-nitrogen stress conditions. A total of 1424 and 1251 metabolites were detected in the two groups respectively using OPLS-DA (orthogonal partial least squares discriminant analysis). Thirteen major metabolites were identified through a search of public databases, including auxin (indole acetic acid, IAA). Eight genes related to auxin signaling were up-regulated by nitrogen stress, detected using quantitative real-time PCR (qRT-PCR). The IAA concentration was elevated in tall fescue leaves after nitrogen stress, detected by enzyme-linked immuno sorbent assay (ELISA). Our research provides valuable information for studying the mechanisms of the response of tall fescue to low nitrogen. The results suggest that application of IAA could be used to alleviate the harmful effects of nitrogen deficiency.