Sulfur is a critical element for plant growth and development, serving as a component of amino acids (cysteine and methionine), iron-sulfur clusters, proteins, glutathione, coenzymes, and auxin precursors. Deficiency or low concentrations of sulfur in the soil can lead to significant growth retardation in plants. The objective of our study was to examine the effects of sulfur (S) deficiency and excess on morphological symptoms, sulfur and nitrogen (N) metabolism, as well as antioxidant activity in soybean. We found that S starvation decreased the fine root length, biomass, and activity, and the chlorophyll content was reduced, while excess sulfur promotes lateral root growth. In contrast to sulfur excess, sulfur deficiency inhibits N and S metabolism levels in both subsurface and above-ground parts, and induced the expression of some sulfur transporters (SULTRs). In this study, we created soybean hairy root lines overexpressing the SULTR gene (GmSULTR2;1a) to observe metabolic changes following sulfur deficiency treatment. The results showed that GmSULTR2;1a saved the sulfur-deficient phenotype, and the antioxidant enzyme activity was much higher than that of the wildtype in the absence of sulfur. Our study revealed the important role of sulfur element in soybean growth and development and the regulation of sulfur deficiency by GmSULTR2;1a.