<p>Subsurface application of organic ameliorant is a novel strategy for stimulating salt leaching out of profile and increasing saline soil fertility and microbial activities. However, it is unclear how microorganisms regulate the contribution of microbial-derived C to SOC sequestration under subsurface organic ameliorant in saline soils. Thus, a 3-year field experiment was done and included three treatments (control, surface organic ameliorant (humic acid (4.5 t ha⁻¹) and manure (30 t ha⁻¹)) at 0–15&#xa0;cm (SF), and subsurface organic ameliorant application at 15–30&#xa0;cm (SSF)) organized into a randomized complete block design. SSF increased soil organic carbon (SOC) stocks at 0–30&#xa0;cm, while SF only affected SOC at 0–15&#xa0;cm. SOC stock at 0–45&#xa0;cm under SF increased by 13% and under SSF by 22% relative to the control. The accumulation of SOC was explained by high fungal necromass C under organic amendments. Random forest and structural equation modeling revealed that increased fungal and bacterial necromass C at 0–30&#xa0;cm soil under SSF and SF is caused by high microbial C use efficiency, the dominance of r-strategist bacteria, and increased fungal richness. The increase of microbial necromass C at 0–30&#xa0;cm under SSF was higher than under SF (27–45 vs 25–37% compared to Control), which was explained by the raised bacterial necromass and its incorporation into mineral-associated organic matter (MAOM). High amounts of root exudates in deep soil under SSF also promoted fungi turnover and accelerated fungal necromass formation at 30–45&#xa0;cm. Overall, subsurface amelioration of saline soils enhanced C sequestration by stabilizing microbial necromass in MAOM.</p> Graphical Abstract <p></p>