Abstracts: Soybean ( Glycine max L.), a vital widely grown grain and oilseed crop in northeastern China, often suffers yield and quality issues from sulfur deficiency due to continuous cropping and its high sulfur demand, resulting in reduced soil sulfur content. This study aimed to enhance soybean soil-available sulfur absorption by inoculating with a clumping arbuscular mycorrhizal fungus (AMF), specifically Funneliformis mosseae, to improve sulfur utilization in the soybean root system and alleviate sulfur deficiency in continuously cropped soybean. The experiment used the soybean variety “Heinong 48” as the test material, and involved four treatments in pots over three years, with soil inoculated with F. mosseae in both non-continuous and continuous cropping systems. Physiological indicators such as soybean mycorrhizal infestation rate, sulfur content, cysteine (Cys), glutathione (GSH), and β-thioglucoside levels were measured. Additionally, functional genes and differential metabolites involved in sulfur metabolism within cysteine and methionine metabolism pathways in soybean roots were analyzed using a combination of transcriptomics and metabolomics methods. The results demonstrated that inoculation with F. mosseae increased Cys, β-thioglucoside, GSH and sulphur contents in soybean roots. It enhanced the enrichment of the cysteine and methionine metabolism pathway and significantly up-regulated the expression of key regulatory genes, such as  ACO1 and  At5g53970, and the sulfur metabolite 5’-methylthioadenosine. Consequently, F. mosseae effectively regulates cysteine and methionine metabolism in soybean roots, promotes root sulfur uptake, and mitigates the sulfur deficiency barrier in continuous soybean cropping. Therefore, this study provides a theoretical basis for applying AMF microbial fungicidesin continuous soybean cropping production.