【Objective】Conservation tillage has attracted increasing attention over recent decades due to its benefits in improving soil quality. A 12-year fixed field experiment was conducted to assess the effects of long-term straw mulching on yields of rice and wheat, soil nitrogen fractions and microbial community with soil layers under a no-till system in Southwestern China.【Method】Two treatments, no-till without straw mulch (NT) and no-till with straw mulch (NTS), were used for the experiment. The soil was sampled at 0-5, 5-10, 10-20, and 20-30 cm soil layers. Soil total nitrogen (TN) and labile nitrogen fractions, including particulate organic nitrogen (PON), microbial biomass nitrogen (MBN), NH4+-N, NO3–-N, and dissolved organic nitrogen (DON) were analyzed. Soil microbial community was determined using phospholipid fatty acid (PLFA) analyses. Crop yields were measured from September in 2013 to May in 2018.【Result】The results showed that compared to the NT treatment, the NTS treatment significantly increased wheat yield by 6.49%, with negligible effects on rice yield. The NTS treatment increased the contents of soil TN at 0-5 cm soil layer, NH4+-N and NO3–-N at 0-5 cm layer, PON at 0-5 and 5-10 cm layers, and DON at 0-5 and 10-20 cm layers but not MBN content. Also, the NTS increased the DON/TN ratio at 0-5 and 10-20 cm layers rather than the ratios of other labile nitrogen fractions to TN. Soil total microbial PLFA and bacterial PLFA were higher in the NTS than in the NT treatment. Additionally, fungal and actinobacterial PLFA were comparable between the two treatments, leading to a lower fungal/bacterial ratio in the NTS. Principal component analysis revealed that the soil microbial community at 20–30 cm layer was separated from the other three soil layers, and soil microbial communities of NTS treatments were generally separated from NT treatments along the first principal component axis. Furthermore, redundancy analysis demonstrated that soil MBN, TN, and ratio of particulate organic carbon to PON were key factors in shaping soil microbial community. Soil TN, labile nitrogen fractions, and microbial PLFA fractions decreased with increased soil layers, irrespective of straw mulching status.【Conclusion】Straw mulching should be recommended to no-till systems in Sichuan province, Southwestern China because it leads to effective improvements in soil nitrogen contents, wheat yields, and soil total microbial PLFA.