Background and Aims <p>Microbial necromass carbon (C) largely contributes to soil organic carbon (SOC) sequestration. Soil microorganisms drive the transformation process of straw C. Crop growth creates an intricate microenvironment for soil microorganisms. However, whether and how cultivating crop affects microbial derived- SOC sequestration in soil profile remains unclear. This research aimed to clarify the effects of cultivating crop on total microbial necromass C (C_M) accumulation, and its contribution to SOC formation, and to explore the factors driving C_M sequestration in soils with straw addition.</p> Methods <p>We conducted an in-situ field experiments with only ¹³C-labeled straw addition (S) and its combined with cultivating crop (SR) in soils with long-term chemical fertilizers application (NP) or without fertilization (CK).</p> Results <p>On the 510th day, straw-derived C_M averagely was 32.1% higher (<i>P</i> &lt; 0.05) in SR versus S treatments and its contribution to newly formed SOC averagely was 13.6% higher (<i>P</i> &lt; 0.05) in NP versus CK treatments. The fungal necromass C (C_F) content at varied soil depths were 15.3%-35.1% lower in NP + SR versus NP + S treatments (<i>P</i> &lt; 0.05) on the 50th day. SR increased the C_F content at 20–40 and 40–50&#xa0;cm soil depths by 23.9% and 49.2%, respectively, relative to S under CK treatments (<i>P</i> &lt; 0.05), while the contrast result was found under NP treatments on the 510th day. Cultivating crop directly and negatively affected the C_M accumulation by regulating microbial biomass and soil properties, which induced the microbial contribution to SOC increased with soil depths during the whole stage.</p> Conclusion <p>The co-existence of cultivating crop with straw addition promoted SOC renewal via newly formed fungal necromass in fertilized soils.</p>