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Subsurface organic ameliorant plus polyethylene mulching is a novel strategy for saline soil improvement and utilization in China. However, little is known about how this strategy impacts soil organic carbon (SOC) both directly (carbon input) and indirectly (soil aggregates protection and microbial regulation). Therefore, a field experiment was arranged and sampled after 3 years, including four treatments, i.e., no ameliorant with and without polyethylene mulching, and subsurface (10–30 cm soil depth) organic ameliorant with and without polyethylene mulching. Subsurface organic ameliorant raised the SOC content in the 0–40 cm by 70% and 90% with and without mulching, respectively. Subsurface organic ameliorant converted the major aggregation size from <0.053 to 0.25–2 mm, indicating that both direct carbon (C) input and indirect soil aggregates protection contributed to the increase in SOC content. In contrast, polyethylene mulching reduced the SOC content by 16% and 6% with and without organic ameliorant respectively. This was due to lower fungal diversity by 9%–14% and supported by the positive effect from fungal diversity to SOC. Further, the subsurface organic ameliorant plus polyethylene mulching increased SOC by 61% through direct C input, indirect soil aggregates protection and microbial regulation. Meanwhile, the contribution of physical protection standardized total effect (STE) = 0.223–0.294] was higher than microbial regulation (STE = 0.040–0.162) according to a structural equation model. Consequently, subsurface organic ameliorant plus polyethylene mulching represents a feasible agricultural practice to increase the SOC content for saline soil amelioration.