Polyamines are small cationic amines required for modulating multiple cell process, including cell growth and DNA and RNA stability. In Salmonella polyamines are primarily synthesized from L-arginine or L-ornithine. Based on a previous study, which demonstrated that polyamines affect the expression of virulence gene in S. Typhimurium, we investigated the role of polyamines in the global gene and protein expression in S. Typhimurium. The depletion of polyamine biosynthesis led to down-regulation of genes encoding structural components of the Type Three Secretion system 1 (TTSS1) and its secreted effectors. Interestingly, Expression of HilA, which is the master regulator of Salmonella Pathogenicity Island 1 (SPI1), was only reduced at the post-transcriptional in the polyamine mutant. Enzymes related to biosynthesis and/or transport of several amino acids were up-regulated, just as the Mg2+-transport systems were three to six-fold up-regulated at both the transcriptional and protein levels. Furthermore, in the polyamine depletion mutant, proteins related to stress response (IbpA, Dps, SodB), were 2–5 fold up-regulated. Together our data provide strong evidence that polyamine depletion affects expression of proteins linked with virulence and stress response of S. Typhimurium. Furthermore, polyamines positively affected translation of HilA, the major regulator of SPI1.