Abstract: The subsoil layer is an important plow layer between topsoil and bottom soil. It is also an important crop root distribution area. However, long-term rotary tillage caused a thick plow pan in the subsoil layer of black soil in Northeast China, which seriously affected the operation of water, fertilizer, gas, and heat in the soil. As is known to all, straw returning is a win-win measure that can not only fertilize the soil but also ensure the utilization of waste resources. Chopped straw is a common way for local farmers to return to the field, while the pelletized straw return is a novel straw return management and is made of straw after highly comminuted and extruded. Our previous research proved that these two forms of deep straw return had a good effect on subsoil physicochemical properties. However, their effects on soil microbial community structure of the subsoil layer were still unknown. To clarified the effect of chopped straw and pelletized straw on soil microbial community structure in subsoil layer of the black soil, then, evaluated the effect of maize straw returning on fertilization of subsoil layer, a one-time deep straw returning experiment was conducted in the black soil of Northeast China from 2016 to 2018. There were five treatments in this experiment, including chopped straw with low dosage (QS1), chopped straw with high dosage (QS5), pelletized straw with low dosage (KL1), pelletized straw with high dosage (KL5), and no straw returning (CK). The contents of soil microbial flora phospholipid fatty acid, soil bulk density, soil water content, pH, soil organic carbon and total nitrogen were measured in the maize harvest season. The results showed that the microbial flora total phospholipid fatty acid content and fungi phospholipid fatty acid content were significantly increased by chopped straw treatments in the first year of straw returning, with a high dosage increasing up to 71.0% and 120.5%. The phospholipid fatty acid content of bacteria, Gram-positive bacteria and Gram-negative bacteria increased more significantly by pelletized treatment, with high dosage treatment up to 41.6%, 29.7% and 26.3%, and the phospholipid fatty acid content of fungi significantly increased in the second year. The phospholipid fatty acid content of each flora, especially the fungal phospholipid fatty acid content of chopped straw with high dosage treatment, was significantly higher than that of pelletized straw with high dosage treatment (21.0%), and only the flora with high dosage straw return had significant changes in the third year. Chopped straw treatment significantly increased the fungi-bacteria ratio in the early stage of straw returning, while low dosage straw returning increased the Gram-positive bacteria: Gram-negative bacteria ratio. With time increasing, the ratio of high dosage straw return increased more significantly, which was conducive to long-term maintenance of ecosystem stability. High dosage of straw returning could significantly change the level of soil physical and chemical factors, which was an important reason for the differentiation of microbial community structure. The significant soil factors changed with the years of straw returning. In the first year, the significant factors were soil bulk density, pH, total nitrogen and C:N ratio (P<0.05), in the second year, the significant factors were soil water content, soil organic carbon, C:N ratio and soil bulk density (P<0.01), and soil organic carbon was the only significant factor in the third year (P<0.05). In conclusion, chopped straw with high dosage treatment had the most obvious difference between microbial community structure and CK, and had a stronger ability to regulate subsoil fungal community. It was more suitable for the improvement of the subsoil fertility of black soil and promoted the resource utilization of straw in Northeast China.