Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present in most living cells. In recent years they have been linked to virulence of several human pathogens including Shigella spp and Salmonella enterica serovar Typhimurium (S. Typhimurium). Central to S. Typhimurium virulence is the ability to survive and replicate inside macrophages and resisting the antimicrobial attacks in the form of oxidative and nitrosative stress elicited from these cells. In the present study, we have investigated the role of polyamines in intracellular survival and systemic infections of mice. Using a S. Typhimurium mutant defective for putrescine and spermidine biosynthesis, we show that polyamines are essential for coping with reactive nitrogen species, possibly linking polyamines to increased intracellular stress resistance. However, using a mouse model defective for nitric oxide production, we find that polyamines are required for systemic infections independently of host-produced reactive nitrogen species. To distinguish between the physiological roles of putrescine and spermidine, we constructed a strain deficient for spermidine biosynthesis and uptake, but with retained ability to produce and import putrescine. Interestingly, in this mutant we observe a strong attenuation of virulence during infection of mice proficient and deficient for nitric oxide production suggesting that spermidine, specifically, is essential for virulence of S. Typhimurium.