Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] is a herbicide widely used in corn production. However, when used under corn-soybean rotation system, it decreases soybean yield. The toxicology mechanisms of atrazine on soybean are rarely studied from the perspective of rhizobia-soybean symbiont formation and nitrogen fixation. In this study, under atrazine stress, high-throughput sequencing of rpoB was performed to evaluate rhizobial diversity in the nodules and soybean rhizosphere, and the correlation between physiological indicators of plant growth and rhizobial diversity was explored. The results revealed that atrazine inhibited soybean growth by disrupting glutamate synthetase activity to reduce total nitrogen (TN) content in the plant. Atrazine altered the nodule structure and reduced leghemoglobin content. High-throughput sequencing of rpoB revealed that the inhibition rate of Afipia genosp. 3 in the nodules under atrazine stress (20 mg·kg⁻¹) was >80 % higher than that in the control (no atrazine). The abundance of Rhizobium leguminosarum and Rhizobium sp. CF142 decreased in the nodules but increased in rhizosphere soil. The abundance of Afipia genosp. 3 and Bradyrhizobium sp. AD1_1_nodule in rhizosphere soil was negatively correlated with TN content and glutamate synthetase activity in soybean. Moreover, soil nitrate nitrogen (NO₃⁻-N) content was the essential factor influencing the rhizobial community diversity under atrazine stress. This study contributed to clarifying the molecular biological mechanism of the effect of atrazine on rhizobia-soybean symbiont formation and nitrogen fixation. Moreover, this study provided new directions and ideas for the subsequent development and application of related prevention and control measures, which can contribute to the more comprehensive development of corn-soybean crop rotation system and ensure the sustainable use of black soil.