Aeromonas hydrophila (A. hydrophila) is an infectious agent that can infect both fish and humans, posing significant threats to aquaculture and public health. Iron acquisition is essential for bacterial survival, colonization, and pathogenicity, as it is crucial to many metabolic and enzymatic processes. Ferrous iron transporter B (FeoB) is a major component of the bacterial iron uptake system, mediating the transport of ferrous iron (Fe²⁺) and playing a key role in cellular iron homeostasis. The present study elucidates the role of FeoB in iron utilization, bacterial physiology, and pathogenicity of A. hydrophila ATCC 7966. A stable feoB-deleted mutant strain (AhΔfeoB) and its complemented strain (CΔfeoB) were constructed. Our results demonstrated that deletion of feoB impaired ferrous‑iron accumulation and substrate affinity, significantly increased sensitivity to clindamycin, sodium deoxycholate, and oxidative stress, reduced biofilm formation, and downregulated biofilm-associated genes. Furthermore, loss of the feoB gene resulted in abnormal cellular morphology and altered expression of 12 out of 15 tested virulence-related genes. Unlike in previous reports, AhΔfeoB showed no significant differences in growth characteristics, hemolytic activity and virulence in a BALB/c mouse infection model under iron-limited conditions. Moreover, DIA-based proteomic analysis revealed that feoB is a core protein essential for iron homeostasis, and its deletion profoundly impacts bacterial metabolism, adaptability, and iron transport. These findings emphasize the central role of FeoB in iron homeostasis and in the regulation of critical physiological functions in A. hydrophila ATCC 7966.