In the present study, we characterized the involvement of Lon protease in bacterial virulence and intracellular survival in Salmonella under abiotic stress conditions resembling the conditions of a natural infection. Wild type (JOL401) and the lon mutant (JOL909) Salmonella Typhimurium were exposed to low temperature, pH, osmotic, and oxidative stress conditions and changes in gene expression profiles related to virulence and metal ion uptake were investigated. Expression of candidate genes invF and hilC of Salmonella Pathogenicity Island (SPI)-1 and sifA and sseJ of SPI-2 revealed that Lon protease controls SPI-1 genes and not SPI-2 genes under all stress conditions tested. The lon mutant exhibited increased accumulation of hydroxyl (OH•) ions that lead to cell damage due to oxidative stress. This oxidative damage can also be linked to an unregulated influx of iron due to the upregulation of ion channel genes such as fepA in the lon mutant. The deletion of lon from the Salmonella genome causes oxidative damage and increased expression of virulence genes. It also prompts the secretion of host pro-inflammatory cytokines leading to early clearance of the bacteria from host cells. We conclude that poor bacterial recovery from mice infected with the lon mutant is a result of disrupted bacterial intracellular equilibrium and rapid activation of cytokine expression leading to bacterial lysis.