The objective of this study was to compare the effects of grazing and non-grazing on the physicochemical properties, stoichiometry, ecto-enzyme activities, and microbial element limitations of soils at five depths in subtropical grassland, China, and to identify the influencing factors of enzyme activities and element limitations. Results showed that grazing, soil depth, and the interaction between grazing and depth remarkably changed soil available phosphorus (AP), available potassium (AK), available calcium (ACa) and available magnesium contents (AMg), bulk density (BD), water content (WC), and β-glucosidase activity (βGC) (p = 6.702e-9 - 0.04739). Compared with no g razing, g razing remarkably declined the 0-5 cm soil AP by 73.10% (p = 0.0250), the 0-5, 5-10, 10-20 cm soil WC by 47.19% (p = 0 .0042), 3 7.19% ( p = 0 .0090), a nd 3 0.80% ( p = 0.0034), however, grazing remarkably increased the 0-5, 5-10, 10-20, 20-30 cm soil ACa by 188.76% (p = 7.9e-05), 93.24% (p = 0.0177) ,84.18% (p = 0.00067), and 38.77% (p = 0.01368), the 0-5, 5-10 cm soil AMg by 186.69% ( p = 0.0016), 78.89% (p = 0.0109), t he 0-5, 5-10, 1 0-20 c m s oil pH b y 0 .51 ( p = 0.0013), 0.37 (p = 0.0006), and 0.27 (p = 0.0114) units, the 0 -5, 5-10, 10-20 cm soil BD by 59.03% ( p = 0.0077), 44.14% ( p = 0.0147), and 35.55% (p = 0.0071), the 30-50 cm soil WC by 22.88% (p = 0.0241), the 0-5 cm soil βGC by 89.49% (p = 0.0011), the 0-5, 5-10 cm soil ACP by 7.87% (p = 0.0300), 6.57% (p = 0.0240), respectively. Grazing exacerbated the microbial C limitation of 0-5 and 10-20 cm soils by 20.51% (p = 0.0078) and 40.38% (p = 0.0209) and switched the soil microbiome from under N limitation to under P limitation at 5-10 cm (p = 0.0390). Specific soil available cations were identified as the important factors that significantly explained the variation of soil ecto-enzyme activities and soil microbial carbon and nutrient limitations. These findings present basic information for the future improvement management of subtropical grassland and understanding the impact of grazing on microbial element limitation.