Salmonella Enteritidis is a major foodborne pathogen that can cause gastrointestinal infections in both humans and animals. As one of the key genes encoding the iron-sulfur cluster assembly, iscA plays a role in the transport of iron ions and energy metabolism. IscA is a conserved A-type iron-binding protein. [Objective] To study the role of iscA in the infection process of Salmonella by constructing an iscA-deleted mutant (ΔiscA) of Salmonella Enteritidis Z11. [Methods] The unmarked in-frame gene deletion method was employed to construct ΔiscA from the laboratory-preserved Salmonella Enteritidis Z11 strain. The wild type (WT) and ΔiscA were compared in terms of motility and biofilm formation. Additionally, the impact of IscA on the virulence of Salmonella Enteritidis was explored in both RAW264.7 cells and a mouse model. [Results] The deletion mutant ΔiscA was successfully constructed. No significant difference in the growth or biofilm formation was observed between ΔiscA and WT, indicating that the deletion of iscA did not affect the normal growth or biofilm formation of Salmonella Enteritidis. However, ΔiscA exhibited a significantly smaller zone of motility than WT at the time point of 6 h, suggesting that the loss of iscA reduced the motility of Salmonella Enteritidis Z11. In RAW264.7 cells, the adhesion and invasion of ΔiscA significantly decreased to 37% and 20%, respectively, of those of WT. Furthermore, the proliferation rate of ΔiscA in the cells was significantly lower than that of WT. Mouse infection experiments revealed that ΔiscA demonstrated reduced colonization in the jejunum and cecum compared with WT. [Conclusion] iscA is closely associated with the virulence of Salmonella Enteritidis. Its deletion affects the motility, adhesion, invasion, and proliferation, ultimately reducing the colonization in the host intestine and influencing the infection process of Salmonella Enteritidis.