Iron deficiency caused by high pH of saline–alkali soil is a major source of abiotic stress affecting plant growth. However, the molecular mechanism underlying the iron deficiency response in cotton (Gossypium hirsutum) is poorly understood. In this study, we investigated the impacts of iron deficiency at the cotton seedling stage and elucidated the corresponding molecular regulation network, which centered on a hub geneGhbHLH121. Iron deficiency induced the expression of genes with roles in the response to iron deficiency, especiallyGhbHLH121. The suppression ofGhbHLH121with virus-induced gene silence technology reduced seedlings’ tolerance to iron deficiency, with low photosynthetic efficiency and severe damage to the structure of the chloroplast. Contrarily, ectopic expression ofGhbHLH121inArabidopsisenhanced tolerance to iron deficiency. Further analysis of protein/protein interactions revealed that GhbHLH121 can interact with GhbHLH IVc and GhPYE. In addition, GhbHLH121 can directly activate the expression ofGhbHLH38,GhFIT,andGhPYEindependent of GhbHLH IVc. All told, GhbHLH121 is a positive regulator of the response to iron deficiency in cotton, directly regulating iron uptake as the upstream gene ofGhFIT. Our results provide insight into the complex network of the iron deficiency response in cotton.