Among the more than 2600 Salmonella serovars, certain serovars can only cause infection in one particular host or a few hosts. Currently, little is known about the underlying mechanisms that contribute to salmonella host-specificity. In this study, we dissected the interactions between avian and cattle macrophage cell lines with the generalist serovar S. Typhimurium, the bovine host-adapted S. Dublin and the avian host-specific S. Gallinarum. Additionally, detailed transcriptome analyses of chicken primary macrophages after phagocytoses of the three serovars were produced and analyzed. Results indicated a poorer invading and survival ability of S. Gallinarum in both avian and cattle macrophages compared to S. Typhimurium and S. Dublin. Furthermore, a higher level of cell death in chicken macrophages was induced by S. Typhimurium and S. Dublin. The transcriptome results showed that S. Typhimurium stimulated a total 625 differentially expressed genes with 443 genes being up-regulated in comparison to none stimulated cells. S. Dublin caused upregulation of 451 genes and downregulation of 205 genes. In contrast, phagocytosis of S. Gallinarum triggered a total of 1114 DEGs, of which 449 genes were specifically regulated only in the S. Gallinarum group. A comparable level of pro-inflammation cytokines and chemokine expression were observed in the S. Gallinarum infection group compared to that of the S. Typhimurium and S. Dublin infection group. Additionally, a specific differential expression of TLR4 and TLR7, both pivotal innate immune receptors, was only observed in S. Gallinarum infection group. A KEGG cluster analysis for unique DEGs in S. Gallinarum infection group indicated that the JaK-STAT signaling pathway was top enriched and thus likely important for the macrophages to respond to S. Gallinarum infection. Taken together, findings presented provide new insights into interaction between Salmonella and its host and increase our understanding of the mechanisms that contribute to S. Gallinarum host specificity.