BACKGROUND & OBJECTIVES: Anaerobic conditions are frequently encountered by pathogens invading the gastrointestinal tract due to low/limiting oxygen conditions prevalent in the small intestine. This anaerobic stress has been suggested to enhance the virulence of gut pathogens. In the present study, we examined the effect of anaerobiosis on the virulence of Salmonella Typhi, a Gram negative bacteria which invades through the gut mucosa and is responsible for typhoid fever. METHODS: Salmonella Typhi (ty2) was cultured in aerobic and anaerobic conditions to compare its virulence by rabbit ileal loop assay, hydrophobicity assay, expression of outer membrane proteins (OMPs) and antioxidant enzymes assay. RESULTS: Anaerobically grown S. Typhi showed significantly higher cell surface hydrophobicity as compared to aerobic bacteria. In vivo toxin production by rabbit ileal loop assay also showed significantly higher fluid accumulation with anaerobic S. Typhi. Expression of OMPs in anaerobic S. Typhi showed a distinct induction of five outer membrane proteins. We observed that exposure of anaerobic S. Typhi to aerobic conditions induced significantly higher level of antioxidant enzymes like superoxide dismutase (SOD) and catalase. INTERPRETATION & CONCLUSION: Our results suggest that exposure of S. Typhi to anaerobic conditions enhances its virulence.