The mitigation mechanisms of a kind of controlled-release nitrogen fertilizer (sulfur-coated controlled-release nitrogen fertilizer, SCNF) in response to O3 stress on a winter wheat (Triticum aestivum L.) variety (Nongmai-88) were studied in crop physiology and soil biology through the ozone-free-air controlled enrichment (O3-FACE) simulation platform and soil microbial metagenomics. The results showed that SCNF could not delay the O3-induced leaf senescence of winter wheat but could enhance the leaf size and photosynthetic function of flag leaves, increase the accumulation of nutrient elements, and lay the foundation for yield by regulating the release rate of nitrogen (N). By regulating the soil environment, SCNF could maintain the diversity and stability of soil bacterial and archaeal communities, but there was no obvious interaction with the soil fungal community. By alleviating the inhibition effects of O3 on N-cycling-related genes (ko00910) of soil microorganisms, SCNF improved the activities of related enzymes and might have great potential in improving soil N retention. The results demonstrated the ability of SCNF to improve leaf photosynthetic function and increase crop yield under O3-polluted conditions in the farmland ecosystem, which may become an effective nitrogen fertilizer management measure to cope with the elevated ambient O3 and achieve sustainable production.