<p>Soil microbial nitrogen (N) immobilizations are important processes of biogeochemical cycles. How the soil N immobilizations change with increasing N inputs, especially in the subsoil, is not clear. Based on a long-term field manipulative experiment in an alpine meadow, we evaluated changes of soil gross NH₄⁺ immobilization rate (GAIR) and NO₃^‒ immobilization rate (GNIR) under six N addition rates at 0–10, 10–20 and 20–40&#xa0;cm soil depths. The corresponding biotic and abiotic mechanisms were also explored. The results showed that GAIR negatively correlated with N addition rate, but GNIR followed the unimodal response (increase first and then drop down) at all the three soil depths. The decrease in substrate supply by mineralization contributed to the decrease of GAIR with increasing N additions at the three soil depths. The changes of substrate supply by nitrification influenced the response of GNIR in the topsoil, but the changes of fungal abundance mediated the responses of GNIR in the subsoil. The increase in GNIR reduced denitrification derived N₂O emission and contributed to retain NO₃^‒, benefitting to the environmental protection. These different responses of GAIR and GNIR to increasing N additions and the different mechanisms underlying the responses from topsoil to subsoil should be considered in biogeochemical models and land management.</p>