Against the background of climate change, grazing accelerates the warming and drying processes in grasslands. There is a relatively clear temperature and humidity difference between grassland used for grazing and grassland that has been excluded from grazing practices. This paper asks whether temperature and humidity differences affect plant roots and cold resistance. Representative plants from an alpine meadow on the eastern margin of the Qinghai-Tibetan Plateau were selected under grazing exclusion and grazing conditions. Dominant plants within and outside of an alpine meadow enclosed for 10 years in the study area were selected as the research objects to study the root morphology and physiological indices of the cold resistance of these plants. The results showed that (1) grazing exclusion (GE) was beneficial for soil temperature and water retention, reduced soil pH, and increased soil nutrient content. Under grazing exclusion conditions, all plant root morphological traits, except root tissue density, increased compared with those under grazing grassland (FG) conditions. Grazed plants adopted resource acquisition strategies, while grazing exclusion plants adopted resource conservation strategies. (2) The changes in the physiological indices of cold resistance in different years and grazing treatments were different. In 2023, the superoxide dismutase (SOD) activity and soluble protein content in GE conditions were significantly lower than those in FG conditions, while the peroxidase (POD) activity was significantly higher than that under FG conditions. The activity of catalase (CAT) in the GE plot was significantly lower than that in the FG plot in 2024. The cold resistance of Gramineae species was lower than that of non-Gramineae plants. A redundancy analysis (RDA) of plant root morphological traits, soil properties, and cold resistance showed that root length and soil pH were the most important factors affecting plant cold resistance. We concluded that grazing exclusion is conducive to plant root growth, but also acidifies the soil and reduces plant cold resistance.