Salmonella infections are among the most common foodborne diseases worldwide. The Enteritidis and Dublin serovars of Salmonella enterica are closely related yet they differ significantly in pathogenicity and epidemiology. Enteritidis is a broad-host-range serovar that commonly causes gastroenteritis and infrequently causes invasive disease in humans. Dublin mainly colonizes cattle but upon infecting humans often results in invasive disease. The aim of this work was to elucidate the molecular factors responsible for the differential pathogenic behavior between both serovars. We performed a quantitative proteomic comparative analysis between one clinical isolate of each serovar grown in vitro under gut mimicking conditions (GMC). Compared to S. Enteritidis, the S. Dublin proteome was enriched in proteins linked to response to several stress conditions, such as those encountered during host infection, as well as to virulence. The S. Enteritidis proteome contained proteins related to central anaerobic metabolism pathways that were undetected in S. Dublin. Similar differences were also found at the transcriptional level, as mRNA levels correlated with proteomic results for 17 of the 20 genes tested in 4 natural isolates of each serovar grown in GMC. This work reveals proteomic differences between two Salmonella serovars with markedly different invasive and host-range characteristics, grown in an infection relevant condition, which were not evident in previous comparative genomic analyses.