The kinetics of extracellular enzymes produced by soil microorganisms regulate soil organic matter decomposition and other ecosystem functions. Atmospheric nitrogen (N) deposition creates a soil environment with elevated reactive N, which is expected to affect the kinetic parameters (Vmax and Km) of hydrolytic and polyphenol oxidative enzymes, as well as soil microbial respiration. We measured the Vmax and Km of seven soil hydrolytic enzymes and polyphenol oxidase (PPO), as well as soil microbial respiration and physiochemical properties, in a temperate steppe after 15 years of multi-level N addition treatments. As the N addition increased, the Vmax of most carbon (C)-degrading and N-degrading hydrolytic enzymes decreased, and this was associated with a decline in soil pH. In contrast, the Vmax of acid phosphatase (AP) increased with increasing N addition. Higher N addition was associated with lower Km of most hydrolytic enzymes, expect for AP and β-xylosidase (BX). There was a quadratic relationship between N addition and the Vmax and Km of PPO, which reached a maximum with the addition of 8g ;N m-2 y-1, and decreased at higher N addition levels. Structural equation modeling indicated that the decline in soil microbial respiration with increasing N deposition was directly mediated by the BG kinetics, due to the fact N-induced acidification negatively impacted the Vmax and Km of BG. Our empirical data on soil enzyme (i.e., Vmax, Km) and its relationship with microbial respiration should be useful for modelling how microbes and substrates interact to regulate soil carbon cycling under N enrichment.