Acidic soil is widely distributed globally. A low soil pH reduces nutrient availability and intensifies the toxicity of metal ions such as aluminum and iron, which is one of the main factors limiting crop yields. This study utilized Arabidopsis thaliana cultivation experiments to investigate the impact of low pH stress on plant root growth and explore the role and molecular mechanism of iron under low pH stress. The results indicated that iron plays a crucial role in the inhibition of root growth under low pH conditions. Under low pH, plants experience excessive iron accumulation in their roots, inducing the generation of large amounts of reactive oxygen species (ROS) such as hydrogen peroxide, causing significant damage to root growth. When iron supply was reduced, this damage was significantly alleviated. Transcriptome analysis revealed that plants activate a broad defense response network in their roots under this stress, including the antioxidant enzyme system to respond to ROS bursts. Metabolic shifts from primary to secondary metabolism occur within the plant, and the expression of the key transcription factor ANAC044 leads to root stem cell death and inhibited root growth. This discovery provides a new perspective for understanding the mechanism of acidic soil harm to plants and offers a theoretical basis for improving crop acid tolerance and rational soil nutrient management.