Antibiotics are important tools used to control infections. Unfortunately, microbes can become resistant to antibiotics, which limit the drugs' usefulness for clinical and veterinary use. It is necessary to improve our understanding of mechanisms that contribute to or enhance antibiotic resistance. Using nalidixic acid (NA) exposure as a sole selective agent, a resistant strain of Salmonella enterica Typhimurium 14028 was derived (2a) that had acquired resistance to chloramphenicol, sulfisoxazole, cefoxitin, tetracycline, and NA. We employed gene array analysis to further characterize this derivative. Results indicate a significant difference (FDR < 5%) in the expression of 338 genes (fold regulation > 1.3) between the derivative and the parent strain growing exponentially under the same conditions at 37°C. Strain 2a showed comparative induction of Salmonella pathogenicity island 2 (SPI2) transcripts and repression of SPI1 genes. Differences in expression were related to efflux pumps (increased expression), porins (decreased expression), type III secretion systems (increased expression), lipopolysaccharide synthesis (decreased expression), motility-related genes (decreased expression), and PhoP/PhoQ and peptidoglycan synthesis (increased expression). It appears that 2a developed altered regulation of gene expression to decrease the influx and increase the efflux of deleterious environmental agents (antibiotics) into and out of the cell, respectively. Mechanism(s) by which this was accomplished or the reason for alterations in gene expression of other genetic systems (curli, flagella, PhoP/PhoQ, and peptidoglycan) are not immediately apparent. Evaluation of transcriptomes within multiple antibiotic-resistant mutants hopefully will enable us to better understand those generalized mechanisms by which bacteria become resistant to multiple antibiotics. Future work in sequencing these genomes and evaluating pathogenicity are suggested.