Salmonella enterica serovar Enteritidis (SE) is a communicable zoonotic bacterium. Macrophages are essential for Salmonella survival, transmission, and infection. In this study, selective capture of transcribed sequences (SCOTS) was used to screen genes preferentially expressed by SE during contact with macrophages from different hosts. We found 57 predicted genes and 52 genes expressed by SE during interaction with avian HD-11 and murine RAW264.7 cells, respectively. These expressed genes were involved in virulence, metabolism, stress response, transport, regulation, and other functions. Although genes related to survival or metabolic pathways were needed during SE infection, different gene expression profiles of SE occurred in the two macrophage cell lines. qRT-PCR results confirmed that most screened genes were upregulated during infection in contrast to the observation during in vitro cultivation, with different expression levels in infected avian macrophages at 2-h and 7-h post-infection. In addition, in vitro and in vivo competition assays confirmed that SEN3610 (a putative deoR family regulator) and rfaQ (related to LPS synthesis) were closely related to SE virulence in both mice and chickens. Three putative transcriptional regulators, SEN2967, SEN4299, and rtcR, were related to SE colonization in mice, while the ycaM mutation caused decreased infection and survival of SE in HD-11 cells without influencing virulence in mice or chicken. Genes showing differential expression between SE-infected avian and murine macrophages indicate specific pathogen adaptation to enable infection of various hosts.