Leptospira interrogans is a zoonotic pathogen found worldwide. Infection with this spirochaete results in leptospirosis, a systemic disease with non-specific febrile symptoms that can progress to multi-organ failure and death. Leptospirosis is a significant burden in developing countries where the mortality rate can reach 20%. Expanding our understanding of leptospiral pathogenesis and the specific mechanisms involved is imperative to the development of vaccines and preventive measures to reduce transmission and disease. The generation of random transposon insertion mutants coupled with high throughput in vivo screening of L. interrogans mutants is facilitating the identification of virulence determinants. A recent screen identified an attenuated htpG mutant. HtpG (High temperature protein G) is the bacterial homologue to highly conserved eukaryotic molecular chaperone heat shock protein 90 (Hsp90). The chaperoning function of HtpG has been associated with stress conditions, including heat and oxidative stress but the role of HtpG in bacterial pathogenesis is unclear. The characterisation of this leptospiral htpG mutant in the hamster model for acute leptospirosis revealed that it is highly attenuated and HtpG is essential for leptospirosis. Complementation of the htpG mutant resulted in complete restoration of virulence. Other bacterial htpG mutants have exhibited sensitivity to heat and oxidative stress. Phenotypic analysis of htpG mutant was performed to determine the cause for attenuation. Sensitivity to heat and oxidative stress were evaluated, along with sensitivity to serum complement, survival with macrophages and protein and lipopolysaccharide expression. HtpG was found to be essential for acute leptospirosis and is a novel virulence determinant of L. interrogans.